Oxygen or glucose deprivation-induced neuronal injury in cortical cell cultures is reduced by tetanus toxin

Neuron,

Vol. 8, 967-973,

May, 1992, Copyright

@ 1992 by Cell Press

Oxygen or Glucose Deprivation-Induced Neuronal Injury in Cortical Cell Cultures Is Reduced by Tetanus Toxin H. Monyer,* R. C. Giffard,+ D. M. Hartley,* M. P. Goldberg,5 and D. W. Chois Department of Neurology and Neurological Stanford University Medical Center Stanford, California 94305

1.1.

Dugan,+ Sciences

Summary We examined glutamate-mediated neurotoxicity in cortical cell cultures pretreated with l-5 &ml tetanus toxin to attenuate the Ca*+-dependent release of neurotransmitters. Efficacy of the tetanus toxin pretreatment was suggested by blockade of electrical burst activity induced by Mg*+ removal and by reduction of glutamate efflux induced by high K+. Tetanus toxin reduced neuronal injury produced by brief exposure to elevated extracellular K+ or to glutamate, situations in which release of endogenous excitatory neurotransmitter is likely to play a role. Furthermore, although glutamate efflux evoked by anoxic conditions may occur largely via Ca2+independent transport, tetanus toxin attenuated both glutamate efflux and neuronal injury following combined oxygen and glucose deprivation. With prolonged exposure periods, the neuroprotective efficacy of tetanus toxin was comparable to that of NMDA receptor antagonists. Presynaptic inhibition of Ca2+-dependent glutamate release may be a valuable approach to attenuating hypoxic-ischemic brain injury. introduction Microdialysis measurements indicate that substantial amounts of glutamate accumulate in the extracellular space during cerebral hypoxia-ischemia in vivo (Benveniste et al., 1984), a key event thought to trigger excitotoxic overactivation of postsynaptic glutamate receptors and subsequent neuronal death (Rothman and Olney, 1987; Meldrum et al., 1987; Choi, 1988). Both decreased cellular glutamate uptake and increased glutamate efflux likely contribute to this pathological glutamate accumulation. The mechanism underlying impaired glutamate uptake is defined: nerve terminals and glia remove extracellular glutamate by energy-dependent processes (Schousboe, 1981) and will fail in this task when high energy phosphate stores become depleted. On the other

*Present address: Department of Molecular Neuroendocrinology, Center for Molecular Biology, Heidelberg, INF 282, 6900 Heidelberg, Germany. + Present address: Department of Anesthesia, Stanford University Medical Center, Stanford, California 94305. t Present address: Department of Endocrinology, Children’s Hospital Medical Center, Boston, Massachusetts 02115. SPresent address: Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110.

hand, several distinct mechanisms could contribute to ischemia-induced glutamate efflux. Ca*+-dependent exocytotic vesicular release from nerve terminals is one attractive possibility, as energy depletion is followed by membrane depolarization and Ca2+ influx through voltage-gated channels. However, studies in cerebrocortical synaptosomes have suggested that the Ca2’-dependent release of glutamate requires energy and thus may be attenuated under hypoxic-ischemic or hypoglycemic conditions (Sanchez-Prieto and Gonzales, 1988). An alternative release mechanism is the Ca2’-independent, carriermediated transport of glutamate from both neurons and glia. The same electrogenic, extracellular Na+dependent carrier normally responsible for removing glutamate from the extracellular space may run in reverse when cells are depolarized and the Na+ gradient is depleted (Knight et al., 1989; Kauppinen et al., 1988); under hypoxic-ischemic or hypoglycemic conditions, it has been proposed to be the major source of glutamate efflux (Nicholls and Attwell, 1990). A third possibility is leakage of glutamate from damaged cells. We have studied the neuronal injury induced by oxygen or glucose deprivation in a simplified model system of murine cortical cell culture and have found that it can be markedly attenuated by N-methyl-oaspartate (NMDA) antagonists (Goldberg et al., 1987; Monyer et al., 1989). Hypoxic neuronal injury in our system (Goldberg et al., 1988) or ischemic injury in vivo (Evans et al., 1988; von Lubitz et al., 1988) can also be attenuated by adenosine and adenosine receptor agonists, agents that block presynaptic glutamate release (Dunwiddie, 1985; Burke and Nadler, 1988). However, adenosine receptor activation may have post- as well as presynaptic actions (Proctor and Dunwiddie, 1987; MacDonald et al., 1986), and the mechanisms underlying adenosine agonist-induced inhibition of neurotransmitter release remain uncertain (Fredholm et al., 1990). We set out to determine whether Ca’+-dependent glutamate release plays an important role in mediating oxygen and glucose deprivation-induced neuronal damage in cortical cell culture. The easiest way of distinguishing experimentally between Ca*+-dependent and Ca2+-independent forms of glutamate release is by manipulating extracellular Ca*+ (or by adding divalent cation antagonists such as Mg*‘). Unfortunately, interpretation of such maneuvers is clouded by the direct reduction of glutamate neurotoxicity induced by these same manipulations (Choi, 1987). As an alternative approach, we turned to a potent natural toxin, tetanus toxin, which has been established to block Ca2+-dependent vesicular transmitter release in several preparations (Habermann and Dreyer, 1986; Simpson, 1986; Penner et al., 1986), without affecting Ca2+-independent, transporter-medi-

P Y

C 40 mV

50

5

I

W+l,

1 min Figure 1. Neuronal Cell Firing Reduced by Tetanus Toxin

induced

by Removal

mM

B of Mg”

IS

(A) Representative penwriter tracing of whole-cell, current-clamp recording from a cortical neuron in physiological salt solution with no added Mg*+. (Band C) Sameas in (A), except that the cultures were pretreated for 24 hr with 3 ngiml tetanus toxin.

ated transmitter Bockaert, 1989).

release

(Van

Vliet

et al., 1989;

Pin and

Results As a first step, we sought evidence that tetanus toxin could interfere with excitatory synaptic transmission in our system. Removal of extracellular Mg2’ has been observed to induce spontaneous excitatory discharges in several in vitro preparations, and it leads to excitotoxic neuronal injury in both hippocampal (Abele et al., 1990) and cortical (Rose et al., 1990) cell cultures. Both discharges and subsequent neuronal death can be abolished by NMDA antagonists. Immediately after removal of extracellular Mg*+, whole-cell current-clamp recording revealed repetitive excitatory discharge bursts in 15 out of,15 cells (Figure IA). When cultures were pretreated with 3 j.rg/ml tetanus toxin for 24 hr, this repetitive discharge activity was markedly attenuated (8 of 15 cells; Figure IB) or completely abolished (7 of 15 cells; Figure IC). Similar results were obtained when cultures were pretreated with 1 or 5 kg/ml tetanus toxin. These treatments were associated with little or no cell injury by examination under light microscopy or efflux of lactate dehydrogenase (LDH) to the bathing medium; higher concentrations were toxic. Another established method for eliciting sustained neurotransmitter release is exposure to high K+. To verify that tetanus toxin pretreatment could attenuate high K+-induced glutamate release in our system, we directly measured evoked excitatory amino acid release to the bathing medium using high-performance liquid chromatography (Figure 2A). Baseline levels of medium glutamate were not altered by pretreatment

45 MIN OXYGEN-GLUCOSE

Figure

2. Tetanus

Toxin

Reduces

60 MIN DEPRIVATION

Evoked

Glutamate

Release

(A) K+-evoked glutamate release. Cultures (control or pretreated with 1 pg/ml tetanus toxin) were exposed to HCSS with either 5.4 or 50 mM KCI for IO min at room temperature. The bathing medium was then analyzed for glutamate by high performance liquid chromatography. Values shown are glutamate concentrations, mean + SEM (n = 3-4 cultures per condition). An asterisk indicates significant difference, p <0.05, from 50 mM KCI control by ANOVA and Student-Neuman-Keul’s test. (B)Clutamate releaseduringcombined oxygen-glucosedepnvation. Cultures (control or pretreated with 1 pg/ml tetanus toxin) were exposed to deprivation of oxygen and glucose, and the medium was sampled after 45 or 60 min. Values represent mean f SEM (45 min, n = 4; 60 min, n = 8). Glutamate levels in shamwashed sister cultures not exposed to oxygen-glucose deprivation were 0.2 + 0.03 WM. Asterisks indicate significant difference from respective controls, p < 0.05, by ANOVA and StudentNeuman-Keul’s test.

with 1 pg/ml tetanus toxin, but this pretreatment produced a significant reduction of the extracellular glutamate accumulation evoked by 50 mM K+. Exposure to 90 mM K+ for 45 min was toxic: cultures developed widespread swelling of neuronal cell bodies and, by the next day, substantial staining of nonviable neurons with trypan blue associated with LDH efflux to the bathing medium (Figure 3). Trypan blue staining (data not shown) and LDH efflux were markedly reduced by addition of IO PM MK-801 ((+)-5-methyl-lo, II-dihydro-5Hdibenzo(a,d)cyclohepten-5,10-iminemaleate) and 10 f.rM CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) to the exposure medium (Figure 3), or 10 PM MK-801 alone (data not shown). Pretreatment

Tetanus 969

Toxin

Reduces

120

120

40

20

0

20

CTRL

MKa

MKP

TnTX

TnTX + MKp

0

CTRL

TNTX

MK-601+

TTX

Figure

aax

Figure 3. Tetanus sure to High K’

Toxin

Attenuates

LDH

Efflux

Following

Expo-

LDH efflux (mean + SEM, n = 3-4 cultures per condition) after exposure to 90 mM K’ under the following conditions: no treatment (CTRL), pretreatment with 1 &ml tetanus toxin for 24 hr (TNTX), 10 uM MK-801 and IO uM CNQX added during the exposure to 90 mM K+(MK-801+ CNQX), or 1 &ml tetrodotoxin (TTX) added during the exposure. In this and subsequent figures, LDH values are determined 1 day after exposure and are scaled to the value present in untreated controls (= 100). Asterisks indicate difference from untreated control at p < 0.05 by ANOVA and Student-Neuman-Keul’s test.

of cultureswith 1 pglrnl tetanustoxin produced partial attenuation of resultant neuronal cell damage (3 of 3 experiments; Figure 3). Unlike the neuronal death induced by MgZ’ removal (Rose et al., 1990), the death induced by high K+ was not prevented by addition of 1 PM tetrodotoxin (Figure 3), suggesting that it was not

mediated by Na+ action potentials. Previous study in hippocampal (Rothman et al., 1987) and cortical (Hartley and Choi, 1989) cultures has demonstrated that the neuronal degeneration induced by brief exposure to high concentrations of glutamate or NMDA can be partially reduced by the late addition of NMDA antagonists after washout of the exogenously added excitotoxin. This posttreatment protective effect suggests that the injury directly attributable to the action of exogenously added excitotoxin is supplemented bythe continued toxic stimulation of NMDA receptors by endogenously released glutamate. Pretreatment of cultureswith tetanus toxin produced a partial reduction in neuronal degeneration following exposure to 500 PM glutamate for 3 min, comparable to the protection offered by 10 VM MK-801 added immediately following NMDA exposure (Figure 4). Notably, tetanus toxin pretreatment combined with 10 PM MK-801 posttreatment produced little or no additional protection compared with 10 FM MK-801 posttreatment alone (Figure 4). Finally, we tested the effect of tetanus toxin pretreatment on the neuronal injury induced by substrate deprivation. Cultures deprived of glucose for 6-8 hr

4. Tetanus

Toxin

Attenuates

Gluiamate

Neurotoxicity

Medium LDH (mean k SEM, n = 4) measured in sister cultures 1 day after exposure to 500 pM glutamate for 3 min alone (CTRL), or with the indicated treatments: 10 uM MK-801 presented acutely during the 3 min glutamate exposure (MKa), 10 uM MK-801 added immediately following glutamate removal (posttreatment, MKp), pretreatment with 1 up/ml tetanus toxin for 24 hr prior to glutamate exposure (TnTx), or pretreatment with 1 ugi ml tetanus toxin combined with posttreatment with 10 uM MK-801 (TnTX + MKp). Asterisks indicate significant difference from untreated control at p < 0.05 by ANOVA and StudeniNeuman-Keul’s test. The conditions MKp, TnTX, and TnTX + MKp were not significantly different from each other; all were significantly different from MKa.

developed neuronal swelling and substantial neuronal degeneration by the following day (Monyer and Choi, 1988). This neuronal degeneration was markedly reduced by addition of 100 PM dextrorphan during exposure and was reduced by about half by pretreatment with l-5 pg/ml tetanus toxin (Figure 5A). Similar neuronal protection was obtained in the presence of tetanustoxin when cell injurywas induced bysimultaneous removal of oxygen and glucose, a severe injury paradigm that causes widespread NMDA receptormediated neuronal death after 45-60 min of exposure (Goldberg et al., 1988, Sot. Neurosci., abstract; Kaku et al., 1991). If the duration of combined oxygen and glucose deprivation was extended to 75 min, the protective effect of tetanus toxin pretreatment was comparable to that of 100 WM dextrorphan (Figure 5B). Tetanus toxin pretreatmentalsosubstantiallyreduced the efflux of glutamate to the bathing medium accompanying45or60minofcombinedoxygenandglucose deprivation (Figure 2B). Discussion The present study suggests that pretreatment of cortical cell cultures with tetanus toxin can partially reduce excitotoxic neuronal degeneration induced by several types of insult: high K+; brief exposure to high concentrations of glutamate; glucose deprivation; and combined oxygen and glucose deprivation. The protective action of tetanus toxin against the first form of excito-

stantial contribution from the continued overstimulation of NMDA receptors by endogenously released glutamate (Hartley and Choi, 1989; Rothman et al., 1987). The source of this endogenously released glutamate is presumably glutamatergic neurons, injured by the initial brief addition of exogenous glutamate. Electrophysiological measurements have indicated that prolonged stimulation of glutamate receptors leaves neurons in a shunted, depolarized state likely

60 ii d

60

ii 40

B

to tax cellular energy stores (Rothman et al., 1987; Sombati et al., 1990, Sot. Neurosci., abstract), and direct measurements of ATP levels in hippocampal cultures exposed to glutamate have confirmed substantial loss of high energy phosphates (Rothman et al., 1987). Since, as noted above, data from cerebral cortical synaptosomes have suggested that energy depletion impairs Ca *+-dependent exocytotic release, we expected most of the glutamate efflux subsequent to excitotoxic exposure to occur by Ca*‘-independent transport or frank leakage. However, the observation that tetanus toxin was as effective as late application of a saturating concentration of the NMDA antagonist MK-801 in reducing neuronal death suggests that a critical portion of this late glutamate efflux may occur via a Ca2+-dependent exocytotic mechanism. The observation that tetanus toxin did not further improve neuronal survival when combined with MK-801 administered after glutamate exposure argues against any substantial postsynaptic action to reduce neuronal vulnerability to glutamate neurotoxicity. MK-801 posttreatment blocks the tox-

120

1T

Figure 5. Tetanus Toxin Substrate Deprivation

Attenuates

Neuronal

Injury

Induced

by

(A) Glucose deprivation. LDH in the media (mean + SEM, n = 3-4) of sister cultures 1 day after a 7 hr exposure to glucose deprivation either alone (CTRL), or with the addition of 100 WM dextrorphan (DX), or with pretreatment for 24 hr with the indicated concentration of tetanus toxin (1 pg/ml). Asterisks indicate difference from untreated control at p < 0.05 by ANOVA and Student-Neuman-Keul’s test. (B) Prolonged glucose and oxygen deprivation. Same as (A), but after a 75 min exposure to combined oxygen and glucose deprivation, an insult severe enough to overcome partially the protective effect of 100 PM dextrorphan (DX). The tetanus toxin-treated condition was statistically different (p < 0.05 by ANOVA and Student-Neuman-Keul’s test) from the dextrorphan-treated condition in (A) but not (B).

toxic degeneration is understandable. Tetanus toxin interferes with the Ca2+-dependent release of neurotransmitters, and here we have specifically demonstrated its ability to reduce both the excitatory discharges induced by Mg2’ withdrawal and the glutamateefflux induced by high K+in our cortical cultures. More surprising is the ability of tetanus toxin pretreatment to reduce neuronai death following brief exposure to high concentrations of glutamate. Earlier experiments suggested that this death reflects a sub-

icity of delayed glutamate release, so injury in this paradigm is largely restricted to that triggered by the 3 min of intense exposure to exogenous agonist. If, for example, tetanus toxin pretreatment down-regulated NMDA receptors or improved neuronal Ca*+ buffering, we would have expected a further reduction in resultant neuronal injury. Equally surprising is the protective action of tetanus toxin against the excitotoxic neuronal damage induced by deprivation of glucose, or oxygen and glucose, two conditions known to produce cellular energy depletion. The majority of glutamate efflux from hypoxic cerebral cortical synaptosomes is Caz+ independent (Kauppinen et al., 1988). It is possible that there might be fundamental differences in transmitter release mechanisms in primary cell cultureand synaptosomal preparations, but such differences need not exist. The neuroprotective effect of tetanus toxin observed here suggests that Ca*+-dependent exocytotic release may play a critical role in the neuronal injury induced by glucose or oxygen deprivation, a suggestion

not

necessarily

incompatible

with

the

idea

that

Ca2+-independent release during energy depletion is large. Ca*-dependent release might occur early in the insult course, while energy stores have fallen enough to compromise membrane potential or Ca*+ homeostasis, but not enough to compromise Ca*+-dependent release. Later on, when energy stores become further depleted, loss of the membrane Na+ gradient

Tetanus

Toxin

Reduces

Hypoxic

Neuronal

Injury

971

may promote reverse operation of the Na’-coupled uptake carrier, leading to the Ca2+-independent extrusion of cytoplasmic glutamate (Nicholls and Attwell, 1990). We cannot exclude the possibility that effects other than those on glutamate release contributed to the observed neuroprotective effects of tetanus toxin treatment. In particular, tetanustoxincan beexpected to reduce release of other transmitters, including y-aminobutyric acid (GABA). However, a reduction in inhibitory tone might be expected to increase not decrease excitotoxic injury. GABA can itself have excitatory actions on young neurons (Cherubini et al., 1991), but we have found direct application of 1 mM CABA to be neither neurotoxic, nor protective against rapidly triggered excitotoxicity in our cultures (Koh and Choi, 1987). Since previous pharmacological studies have suggested that overactivation of glutamate receptors is the key event underlying the neuronal death in the paradigms studied here, the documented ability of tetanus toxin to reduce glutamate efflux is a sufficient explanation for neuroprotection. There may be several reasons why even a small Ca2+-dependent component of glutamate release might be disproportionately important in mediating subsequent neuronal injury. For example, the location of vesicular release might be better suited to stimulate postsynaptic receptors than the location of carrier-mediated release. Or, given the potential positive-feedback natureof excitotoxic injury(injured neurons damaging other neurons), early release may be responsible for initiating a widening series of subsequent destructive cascades, and thus blockade of early release might abolish a large amount of downstream glutamate release and neuronal destruction. Tetanus toxin-induced reduction in extracellular glutamate accumulation during oxygen and glucose deprivation might reflect lowering of both a small Cal+dependent component, and a larger subsequent Ca2+-independent component. Finally, the toxicity of glutamate released priorto complete neuronal energy depletion might be greater than the toxicity of the same amount of glutamate released after complete energy depletion, as postsynaptic energy depletion may reduce the activation of NMDA receptors (Mody et al., 1988). Furthermore, Ca2’-independent glutamate release may also be critical for neuronal damage. Indeed, the incomplete nature of the protective effect provided by tetanus toxin against shorter durations of glucose or oxygen-glucose deprivation most likely reflects the occurrence of injury due to tetanus toxin-insensitive, Ca2+-independent glutamate release; NMDA antagonists produce nearly complete neuroprotection in these paradigms involving shorter insult periods (Monyer et al., 1989; Kaku et al., 1991). Alternatively, the ability of tetanus toxin to block Ca*+-dependent glutamate release may not be complete. It will be desirable to explore further thecontribution of Ca2+-in-

dependent release when methods for selectively blocking it become available. While the neuroprotective effect of tetanus toxin was incomplete, it was at least comparable to that produced by MK-801 in the setting of prolonged insult durations. The well-maintained nature of tetanus toxin neuroprotection may be explained by an ability to reduce AMPAlkainate receptor-mediated injury concurrent with reduction of NMDA receptor-mediated injury (Kaku et al., 1991). Other measures directed at reducing the pathological efflux of glutamatefrom presynapticterminalsorothersourcesmay share this favorable profile. Experimental

Procedures

Cortical Cell Culture Cultures of mixed neocortical neurons and glia were prepared as previously described from embryonic day 15-17 fetal mice (Choi et al., 1987). Briefly, dissociated neocortical cells were plated on modified polystyrene (Falcon Primaria) 15 mm culture wells and 35 mm dishes in Eagle’s minimal essential medium (MEM, Earle’s salts, supplied glutamine free) supplemented with glucose (total 21 mM),glutamine (2 mM), 10% equine serum, and 10% fetal bovine serum; this medium contained about 0.8 mM Mg2+. Nonneuronal cell division was halted when the glial layer was confluent (after IO-14 days in vitro), by a 24-72 hr exposure to 10 NM cytosine arabinoside. For some experiments, cortical cells were plated upon a previously established monolayer of cortical glia, and cytosine arabinoside was added after 5-7 days. Cultures were selected for study after 13 days in vitro. Tetanus toxin was provided by the Department of Public Health, Commonwealth of Massachusetts, and stored at -7OOC until use. Electrophysiology Dishes were maintained on the stage of an inverted Nikon Diaphot microscopeat room temperature. We recorded from single neurons in the whole-cell, current-clamp configuration. The pipette solution contained 140 mM KCI, 10 mM HEPES, 5 mM MgATP, 5 mM EGTA, and 0.5 mM CaCI, (titrated to pH 7.3 with NaOH). The bath solution had the following composition: 140 mM NaCI, 2.8 mM KCI, 1 mM CaCl?, 10 mM HEPES, 5 mM glucose, 0.005 mM glycine (pH adjusted to 7.3 with NaOH). Excitatory Amino Acid Exposure Exposure to glutamate was carried out at room temperature in a HEPES-buffered control salt solution (HCSS), substituted for culture medium by triple exchange. After 3 min, the neurotoxin was washed out and replaced with MEM with augmented glucose (21 mM), and the cells were returned to the incubator. Neuroprotective drugs were added either 24 hr before this excitotoxic exposure (pretreatment), during the exposure (acute), or immediately after the exposure (posttreatment). In the latter case, the drug remained in the medium until the next day, when neuronal injury was assessed. Glucose and Combined Oxygen-Glucose Deprivation Glucose deprivation was carried out by exchanging the culture medium for a defined solution resembling MEM, but lacking glucose and vitamins. After glucose deprivation for 6-8 hr at 37”C, exposure was terminated by adding a small amount of concentrated glucose to the exposure solution (final concentration 5.5 mM). Cultures were returned to the incubator until the next day when injury was assessed. Combined oxygen and glucose deprivation was carried out by exchanging the culture medium with a deoxygenated, bicarbonate buffered salt solution (02 partial pressure < 2 mm Hg), identical to Earle’s balanced salt solution except for the lack of glucose. Cultures were kept for 45-75 min at 37OC in a humidified atmo-

sphere ot Nz with 5% CO,. This insult was terminated by a thorough washout of the exposure medium with oxygenated MEM containing 5.5 mM glucose. The cultures were then returned to a normoxic, humidified incubator (37”C,5% COz)overnight prior to assessing injury. Assessment of Neuronal Cell Injury Overall neuronal cell injury was estimated in all experiments by examination of cultures with phase-contrast microscopy at 100 x to 200x. In some experiments, this determination was verified by subsequent bright-field examination of trypan blue staining, a dye stainingdebrisand nonviablecells. Overall neuronal injury was further determined in a quantitative fashion in most experiments by the measurement of LDH, released by damaged or destroyed cells into the extracellular fluid 1 day after the experiment, at which point the process of cell death was largely complete. A small amount of LDH present in the medium from sister cultures exposed to sham-wash conditions was subtracted from all totals. High Performance Liquid Chromatography Amino acid accumulation in the extracellular medium was measured by reversed-phase high performance liquid chromatographywith o-phthaldialdehyde (OPT) precolumn derivitization and fluorescence detection (Lindroth and Mopper, 1979). Aliquots (30 ~1) were mixed with 30 ~1 of OPT, 2-mercaptoethanol reagent (Sigma) and injected onto a 3.9 x 150 mm Cl8 analytical column (Resolve, Waters). Glutamate and aspartate were separated from each other and from other amino acids using a rapid gradient elution (flow rate 1.5 ml): solvent A (0.1 M potassium acetate [pH 7.41, methanol, 80:20) to solvent 6 (potassium acetate, methanol, 20:80) over 3 min. OPT derivatives of glutamate and aspartate were identified by their retention times, and concentrations were quantified by peak area comparisons to a standard solution containing 40 amino acids (Sigma). Acknowledgments

Choi, D. W. (1988). Glutamate neurotoxicity nervous system. Neuron 7, 623-634.

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Choi, D. W., Maulucci-Cedde, M., and Kriegstein, A. R. (1987). Glutamate neurotoxicity in cortical cell culture. J. Neurosci. 7, 357-368. Dunwiddie, the central

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Evans, M. C., Swan, J. H., and Meldrum, B.S. (1988).An analogue, 2-chloroadenosine, protects against long opment of ischaemic cell loss in the rat hippocampus. Lett. 83, 287-292.

In

adenosine term develNeurosci.

Feldholm, B. B., Duner-Engstrom, M., Fastbom, J., Hu, P.-S., and van der Ploeg, I. (1990). Role of G proteins, cyclic AMP, and ion channels in the inhibition of transmitter release by adenosine. Ann. NY Acad. Sci. 604, 276-288. Goldberg, M. P., Weiss,J. W., Pham, P. C., and Chow, D. W. (1987). N-methyl-o-aspartate receptors mediate hypoxic neuronal injury in cortical culture. J. Pharmacol. Exp. Ther. 243, 784-791. Goldberg, Adenosine or glucose

M. P., Monyer, H.,Weiss, J. W.,and Choi, D. W. (1988). reduces cortical neuronal injury induced by oxygen deprivation in vitro. Neurosci. Lett. 89, 323-327.

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Hartley, D. M., and Choi, D. W. (1989). Delayed rescue of N-methyl-u-aspartate receptor-mediated neuronal injury in cortical culture. J. Pharmacol. Exp. Ther. 250, 752-758. Kaku, D. A., Goldberg, M. P., and Choi, D. W. (1991). Antagonism of non-NMDA receptors augments the neuroprotective effect of NMDA receptor blockade in cortical cultures subjected to prolonged deprivation of oxygen and glucose. Brain Res. 554, 344347. Kauppinen, R. A., McMahon, H., and Nicholls, D. G. (1988). Ca?‘dependent and Ca>+independent glutamate release, energy status and cytosolic free Cal+ concentration in isolated nerve terminals following metabolic inhibition: possible relevance to hypoglycemia and anoxia. Neuroscience 27, 175-182.

We thank K. Rose for expert assistance. This research was supported by NIH grant NS26907 to D. W. C. Postdoctoral support to H. M. was provided by the Deutsche Forschungsgemeinschaft. R. G. G. was supported by NIH grant NS01425 and an AnesthesiaYoung Investigator/FAER Investigator Award from the Foundation for Anesthesia Education and Research. M. P. G. received support from a Dana Foundation Fellowship and a grant from the Brain Trauma Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact.

Koh, J., and Choi, D. W. (1987). Effect of anticonvulsant drugs glutamate neurotoxicity in cortical cell culture. Neurology 319-322.

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MacDonald, R. L., Skerritt, J. H., and Werz, M. A. (1986). Adenosineagonists reducevoltage-dependentcalcium conductanceof mouse sensory neurones in cell culture. J. Physiol. 370, 75-90.

December

17, 1991; revised

February

21, 1992.

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Address correspondence to Dennis W. Choi, MD, ington University School of Medicine, Department ogy,Box8111,660SouthEuclidAvenue,St.Louis,Missouri63110.

PhD, Washof Neurol-