Glucocorticoids increase extracellular [3H]d -aspartate overflow in hippocampal cultures during cyanide-induced ischemia

BRAIN RESEARCH ELSEVIER

Brain Research 654 (1994) 8-14

Research report

Glucocorticoids increase extracellular [3H]D-aspartate overflow in hippocampal cultures during cyanide-induced ischemia Yun-Chia Chou **, Walter J. Lin, Robert M. Sapolsky * Department of Biological Sciences, Stanford Unit:ersity, Stanford, CA 94305, USA Accepted 26 April 1994

Abstract

Glucocorticoids (GCs), the adrenal steroid hormones secreted during stress, exacerbate neuronal death in the hippocampus during ischemia. Since ischemic brain damage is ascribed to an elevated level of extracellular excitatory amino acids (EAAs), this study was undertaken to investigate the effect of GCs on EAA homeostasis in hippocampal cell cultures during the insult of cyanide exposure. Using o-[2,3-3H]aspartic acid ([3H]D-Asp) as a tracer, we found that corticosterone (CORT, the physiological GC in rats) increased the accumulation of extracellular [3H]D-Asp by 25% in hippocampal cultures during cyanide-induced ischemia. CORT had no effect on the release of [3HID-Asp. Instead, analysis of [3H]D-ASp uptake kinetics indicates that CORT decreased the maximum uptake rate and the Michaelis constant by 44% and 50%, respectively, in cells treated with cyanide. It is concluded that, during cyanide-induced ischemia, CORT might enhance extracellular overflow of [3HID-Asp by decreasing its uptake, thereby endangering neurons. Key words: Glucocorticoid; Ischemia; Hippocampus; Cell culture; Cyanide; [3H]o-Aspartate

I. Introduction

Much evidence has shown that shortage in brain energy supply, as occurs during ischemia, increases the overflow of excitatory amino acids (EAAs) such as glutamate (Glu) and aspartate (Asp) [2,7,18,20]. These excessive EAAs Would activate N-methyl-D-aspartate (NMDA) receptors, raising levels of intracellular calcium (Ca 2+). Such Ca 2÷ may generate oxygen radicals and activate catabolic enzymes which lead to neuron death. Thus, an understanding of factors which modulate this E A A / N M D A / C a 2÷ cascade will greatly improve our ability to decrease brain damage during ischemia. One important modulator of toxicity appears to be glucocorticoids (GCs). GCs, adrenal steroid hormones secreted during stress, disrupt glucose transport and metabolism in hippocampal cells [17,22,25,56] and thus render them less capable of withstanding neurological

* Corresponding author. Fax: (1) (415) 723-6132. ** Present address: Institute of Physiology, National Yang-Ming Medical College, Taipei, Taiwan, ROC. 0006-8993/94/$07.00 © 1994 Elsevier Science B,V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 5 3 5 - K

insults such as ischemia [21,28,33,50], seizure [47,54], exposure to antimetabolites [47], to cholinergic [1,24] or serotonergic toxins [23]. During excitotoxic seizure, GCs increase the overflow of Glu in the hippocampus [52]; furthermore, the GC endangerment of hippocampal neurons is NMDA-receptor-dependent [3]. Moreover, GCs raise the level of intracellular Ca 2+ and thus enhance Ca2+-dependent degenerative events (such as cytoskeletal degradation) [12-14]. All these events, however, are prevented by energy supplementation [12-14,52,55]. Taken together, it appears that GCs may endanger hippocampal cells by impairing energy homeostasis and thus exacerbating the E A A / N M D A / C a 2+ cascade. As noted, GCs increase the accumulation of extracellular EAAs in the hippocampus during excitotoxic seizure [52] and in response to stress [29,32]. This study was undertaken to test whether the same occurs in hippocampal cultures during ischemia and to explore the mechanism(s) by which GCs affect EAA trafficking - - do GCs enhance the release of these EAAs a n d / o r disrupt their uptake? Using [3HID-Asp) as a tracer, our data suggest that, during cyanide-induced ischemia, GCs impair the uptake of [3H]D-Asp and thus increase

Y.-C. Chou et al. /Brain Research 654 (19941 8-14

the overflow of the amino acid in mixed hippocampal cultures.

9

Table 2 Release of [3H]D-Asp from mixed hippocampal cultures in K R P H S buffer containing 5 m M glucose (control) or 5 mM glucose, 55 m M KCI and 1.3 m M CaCI 2 (KCI/CaCI 2) Experimental condition

[3H]D-ASp release (as % of control)

2.1. Primao' hippocampal cultures

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Hippocampal cultures were prepared from Sprague-Dawley rats as described elsewhere [22,55]. In brief, hippocampi were dissected from fetal rat brains (17-18th day of gestation) and treated with 0.05% trypsin (Gibco, NY). The dissociated cells were then washed and suspended in Dulbecco's modified Eagle medium (DMEM; Gibco, NY) supplemented with 10% horse serum (Hyclone Laboratories, Logan, UT). Cells were then plated in 12- or 24-well plates precoated with poly-D-lysine (Sigma, MO) at a density 5 × 1 0 6 c e l l s / c m 2 and incubated at 37°C in a humidified incubator with 5% CO 2 / 9 5 % O~. Cultures were used at 10-13 days of age, at which time 5(J-70% of the cells were astrocytes [22].

The release of [3HID-Asp from cultured cells was monitored in fractions collected from the superfusion chamber over a period of 40 min and 30 s as described in section 2. Peaks of [3H]D-Asp released from cultures were quantitated as percentages of basal release of [3H]D-Asp. Data are expressed as percentages of peak release in control groups (22.38 p m o l / m g p r o t e i n / m i n , 30.93 p m o l / m g prot e i n / m i n and 19.91 p m o l / m g protein/min). Values represent m e a n s + S.E.M. of three independent experiments (6 wells). a Indicates significantly different at P < 0.05 from control groups.

2. Materials and methods

2.2. Studies of [-¢H]P-Asp accumulation The experiments were carried out as described by Yu et al. [57] with the following modifications. Cultured cells grown in 12-well plates were treated with 0 or 1 # M of corticosterone (CORT; the physiological GC in rats) in D M E M at 37°C for 24 h. Cells were then washed twice with K R P H S (136 m M NaCI, 4.7 m M KCI, 1.25 m M MgSO 4, 1 m M CaCI2, 5 m M NaH2PO4, 20 m M HEPES, pH 7.40) buffer containing 5 m M glucose. Subsequently, cells were loaded with 0.5 M [3H]D-Asp (12.8 C i / m m o l , D u P o n t New England Nuclear, MA) in K R P H S buffer containing 5 m M glucose and 2 m M glutamine at 37°C for 30 min. D-Asp is not metabolized and labels the transmitter pool in a fashion similar to Glu [6,11]. Afterwards, cells were washed and incubated with 650 liters of corresponding buffer (KRPHS buffer containing indicated concentrations of glucose, sodium cyanide, C O R T , CaC12 and KCI) at 29°C. An aliquot of 20(1 #1 was collected immediately. Subsequently, the radioactivity was washed out by replacing 400 #1 of medium at regular intervals of 45 s for 5 min 15 s and at intervals of 5 min for another 25 min. Cells were then lysed with 1 N NaOH. The radioactivity of each 'wash-out" aliquot was counted in a liquid scintillation spectrometer and the amount of [3H]b-Asp accumulated at the end of each wash-out period was determined. Thus, the summation of [3H]D-Asp accumulated at each wash-out period indicates the total radioactivity accu-

mulated over the incubation period. Furthermore, the summation of [3H]o-Asp in all the wash-out samples and [3H]D-Asp remaining in cell lysates indicates the total radioactivity taken up by cultured cells at the beginning of the experiment.

2.3. Studies of [~H]o-Asp release Release of [3H]D-Asp from hippocampal cultures was measured with modifications of the methods of Belhage et al. [6]. Cultured cells were treated with C O R T and [3H]D-Asp as described above. After two rapid washes with K R P H S buffer containing 5 mM glucose, each

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Table 1 Accumulation of [3H]o-Asp in the extracellular media in mixed hippocampal cultures following exposure to K R P H S buffer containing 5 m M glucose (control) or 5 m M glucose, 55 m M KCI and 1.3 m M CaCI z (KC1/CaC121 Experimental condition Control KCI/CaCI 2

[3H]D-ASp accumulation (as % of control) 1(/(I ± 8 128 ± 6 a

The amount of [3H]D-Asp accumulated in the m e d i u m over a period of 30 rain and 15 s was measured as detailed in section 2. Data are expressed as percentages of extracellular [3H]D-Asp accumulation in control groups. The levels of extracellular [3HID-Asp in control groups represent the basal a m o u n t s of [3H]D-Asp accumlated over the experimental period (458.54 p m o l / m g protein, 475.12 p m o l / m g protein and 585.5 p m o l / m g protein, n = 3). Values represent m e a n s _+S,E.M. of three independent experiments (6 wells). Indicates significantly different at P < 0.05 from control groups.

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Fig. 1. Accumulation of [3H]D-Asp in the extracellular media following 30 min exposure to K R P H S buffer containing 5 m M glucose (control), 2 m M sodium cyanide (CN), 5 m M glucose plus 1 /zM C O R T ( C O R T ) or 2 m M sodium cyanide plus l p,M C O R T ( C O R T / C N ) . The a m o u n t of [3H]D-ASp accumulated in the medium during the experimental period was quantitated as described in section 2. Data are expressed as percentages of extracellular [3H]DAsp in control groups (560.56 p m o l / m g protein, 388.14 p m o l / m g protein and 430.95 p m o l / m g proteins). The columns represent averages of 10 wells (3 experiments) with S.E.M. indicated as vertical bars. * Indicates significantly different at P < 0.05 from all 3 other groups.

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well was covered tightly with a plunger with two tubings, allowing the inflow and outflow of superfusion buffer, respectively. The superfusion buffer was delivered by a peristaltic pump (0.5 ml/min) and collected in a fraction collector (0.5 rain/fraction). Since all the release experiments were performed in this continuous superfusion system, the possibility of reuptake of released [3H]o-Asp is considered very low. Cells were first superfused with KRPHS buffer containing 5 mM glucose at 29°C for 10 rain, allowing to measure the basal release of [3H]D-Asp. An ischemia-like condition was induced by changing the buffer to KRPHS buffer containing 2 mM sodium cyanide [19,31,43-46]. KRPHS buffer containing 55 mM KcCI(with a reduction of equimolar NaC1) and 1.3 mM CaCI 2 was used to test the Ca2+-dependency of [3H]D-Asp release [6,11,43]. Buffer was collected at 30-s intervals and radioactivity in each fraction was counted.

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Measurement of [3H]D-Asp uptake was performed according to Drejer et al. [11] and Virgin et al. [57] with some modifications. Cultured cells grown in 24-well plates were treated with CORT as described. Subsequently, cells were washed with KRPHS buffer twice and preincubated for 30 min at 37°C with 0 or 1 /xM CORT in KRPHS buffer containing 2 mM sodium cyanide. [3H]D-Asp (final concentrations ranging from 5 to 60 txM) was added and uptake experiments were carried out for 5 min at 29°C. In addition, a separate set of cells were incubated with [3HID-Asp at 0°C for 30 s to determine the amount of [3H]D-Asp entrapped in culture plates. Uptake was terminated by removal of medium followed by three rapid washes with ice-cold KRPHS buffer [57]. Cells were then lysed with 1 N NaOH and lysates were used for counting and protein

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Fig. 3. Representative Lineweaver-Burk plots of initial [3HID-Asp uptake rates in hippocampal cultures treated with 0 (o) or 1 /xM ( v ) CORT during cyanide-induced ischemia. The lines repre~nt Lineweaver-Burk plots and are fitted to the experimental values by weighted regression analysis (Segel, 1975). The y-axis represents reciprocal velocities of [3H]D-Asp uptake and each point is the mean value obtained from 3 wells with S.E.M. indicated as vertical bars. Vmax and K m values were determined by linear regression analysis of the Lineweaver-Burk plots, based on the equation V= Vma~[DAsp]/{K m + [D-Asp]} in which V indicates velocities, and [D-Asp] the concentration of [3H]D-Asp. The Vmax values were t2.26 and 8.65 nmol/mg/min, respectively for cultures treated with 0 and 1 M CORT during cyanide-induced ischemia. The K m values were 21~26 and 11.57 #M, respectively for cultures treated with 0 or 1 IzM CORT during cyanide-induced ischemia. * Indicates significantly different at P < 0.05 from CN groups.

1 O0 determination. Specific [3H]o-Asp uptake was obtained by subtracting the entrapped [3HID-Asp from the total uptake. The kinetic parameters of uptake, the maximum uptake rate (Vmax) and the Michaelis constant (Km), were determined as described previously [10,57].

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CONTROL CORT CN CORT/CN Fig. 2. Peaks of [3H]D-Asp released in KRPHS containing 5 mM glucose (control) or 2 mM sodium cyanide (CN), 5 mM glucose plus 1 IzM CORT (CORT) or 2 mM sodium cyanide plus 1 /xM CORT (CORT/CN). The release of [3HID-Asp from cultured cells was analyzed in fractions collected from the superfusion chamber during the experimental period as described in section 2. Peaks of [3HID-Asp released from cells were quantitated as percentages of basal release of [3H]o-Asp. Data are expressed as percentages of peak release in control groups (24.03 pmol/mg protein/min, 19.23 pmol/mg protein/min and 39.05 pmol/mg protein/min). The columns represent averages of 9 welis (3 experiments) with S.E.M. indicated as vertical bars.

Data were analyzed by paired Student's t-test or by analysis of variance (ANOVA) followed by a Fisher PLSD or Scheffe F-test. All data are expressed as mean + S.E.M.S.E.M. values for control groups are obtained by expressing each control value as a percentage of the mean control value.

3. Results As validation of our culture system, we observed a s i g n i f i c a n t i n c r e a s e in t h e a c c u m u l a t i o n o f e x t r a c e l l u l a r [ 3 H ] D - A s p in r e s p o n s e t o t h e s t i m u l i o f C a 2+ a n d K C l - i n d u c e d d e p o l a r i z a t i o n ( T a b l e 1), c o n s i s t e n t w i t h p r i o r r e p o r t s [43]. M o r e o v e r , C a 2+ a n d KC1 t o g e t h e r e n h a n c e d [ 3 H ] D - A s p r e l e a s e ( T a b l e 2), in a g r e e m e n t

Y.-C. Chou et a l . / Brain Research 654 (1904) 8 - 1 4

with numerous reports of the calcium-dependency of such release [6,36,43]. A cyanide dose was chosen which was insufficient to stimulate extracellular [3H]D-Asp accumulation. As described in section 2, 'accumulation' represents the combination of release and uptake. As shown in Fig. 1, the accumulation of [3H]D-Asp in cyanide plus C O R T - t r e a t e d wells was significantly higher than that in cells treated with cyanide alone. C O R T alone did not affect extracellular [3H]D-Asp accumulation. To investigate the mechanism(s) underlying this phenomenon, we then examined whether C O R T exacerbated cyanide-induced [3H]D-Asp release in hippocampal cultures. As described, 'release' experiments were done with a superfusion system which eliminated the possibility of reuptake of [3H]D-Asp, thus yielding a pure measure of relase. As shown in Fig. 2, cyanideplus-CORT-treated wells did not have higher release rates than did cyanide-treated alone. We then tested the possibility that C O R T decreased [3H]i>Asp uptake in hippocampal cultures during cyanide-induced ischemia. Fig. 3 shows the Lineweaver-Burk plots of initial [3H]D-Asp uptake rates in hippocampal cultures treated with 0 or 1 /xM C O R T during cyanide-induced ischemia. Analysis of the data revealed a high-affinity uptake system following Michaelis-Menten kinetics, and thus the maximum uptake rate (V~,×) and the Michaelis constant ( K m) were determined. As shown in Fig. 4, C O R T decreased the I/,.,.~ and the K m values by 44% and 50%, respectively, during cyanide-induced ischemia.

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4. Discussion

Numerous studies have demonstrated that GCs exacerbate neuron loss in the hippocampus and in hippocampal cultures during ischemia [21,28,33,4850,54,55]. In view of the pathogenic mechanisms of ischemic brain damage, we hypothesized that GCs exacerbate the damaging cascade of EAAs during the insult. In this study, we observed that C O R T increased the overflow of [3H]D-Asp in hippocampal cultures during cyanide-induced ischemia. Furthermore. our data indicate that this is most likely due to C O R T inhibition of D-Asp uptake, rather than enhancement of its release. Before considering these findings, it is important to discuss the rationale for using D-Asp rather than GIu. Given the extensive metabolism of Glu in brain cells [15], D-Asp (a non-metabolizable Glu analogue) was chosen to label the transmitter pool of GIu. as suggested previously {5,6,9,11]. While the use of D-Asp has been questioned, since it is not accumulated into isolated synaptic vesicles {30,35,36,42], D-Asp is taken up by putative Glu neurons in brain [4,5,9]. Moreover, upon depolarization, Ca 2+ triggers the release of [3HID-Asp from glutamatergic terminals [4-6,9,11] (Table 2). Taken together, it appears that [3H]D-Asp gains access to the transmitter pool of Glu, and thus was used to infer its release and uptake. Ample evidence indicates that ischemia ew)kes massive release of EAAs which in turn leads to neuron loss [7,11,20]. In the present study, we chose a subthreshold

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dose of cyanide which was insufficient, on its own, to cause significant [3HID-Asp accumulation in mixed hippocampal cultures. However, the same cyanide dose caused significant accumulation in cultures treated with CORT, suggesting this as a mechanism by which C O R T might accelerate the damaging cascade of EAAs during cyanide-induced ischemia. Levels of extracellular EAAs are determined by a balance between E A A release and removal [37]. In mixed hippocampal cultures, C O R T had no effect on the release of [~H]D-Asp during cyanide-induced ischemia. In contrast, C O R T influenced the dynamics of [3H]l>Asp uptake. As one manifestation of this, C O R T reduced the maximal uptake rate of [3H]D-Asp, suggesting that C O R T prolonged the process of [3H]t)-Asp removal by uptake carriers (i.e. [3H]D-Asp would occupy the carrier for a longer time) [51]. With the uptake of each E A A molecule, three Na ~ ions (or perhaps two Na + and one H +) are co-transported into the cell, whereas one K + ion is counter-transported out of the cell. Thus, the uptake system of E A A requires energetically costly maintenance of electrochemical gradients [15,37]. In support of this view, glial uptake of Glu fails with energy deprivation [27,53,57]. Due to their disruptive energetic effects, C O R T may impair such gradients, thereby slowing down [3Hit>Asp uptake and hence decreasing the effective number of uptake carriers. Surprisingly, C O R T also decreased the K m values under 'ischemic' conditions. This is seemingly at odds with our prior report of C O R T increasing K m values of Glu uptake in cultured hippocampal astrocytes [57]. The difference in cell types (mixed cultures vs. pure astrocyte glial) might be of importance, In addition, the magnitude of the effect of C O R T on K,,~ values in that study decreased as cells were cultured in lower glucose concentrations, and the present study was carried out in the absence of glucose during cyanide-induced ischemia. The combined effects of C O R T on Vm~× and K m values make [3H]D-Asp uptake a higher affinity, yet lower capacity system. Functionally, this predicts that under conditions of the highest synaptic concentration of EAAs (i.e. during an excitotoxic insult), C O R T will decrease the capacity of cells to remove the EAAs from the synaptic cleft. Taken together, our data indicate that, during cyanide-induced ischemia, C O R T decreased the uptake of [3H]D-Asp and thus enhanced the extracellular overflow of this amino acid in mixed hippocampal cultures. Growing evidence indicates that the release of Glu during anoxia/ischemia is Ca-' +-independent as a result of a reversal uptake system for Glu [1 1,37]. In this context, it is possible that such an uptake system may become crucial in terms of provoking massive overflow of extracellular EAAs during ischemia. Accordingly, this system may become more sensitive to energetic disruption as evidenced by our finding that

C O R T reduced [3H]D-Asp uptake while having no effect on the release of the amino acid. Cerebral ischemia leads to a dramatic rise in GC concentrations in humans [8,41], and these elevated GC levels are associated with greater mortality and poorer functional outcome [16,34,38-40]. The present study demonstrates that GCs enhance the accumulation of extracellular EAAs. Although this study did not explicitly test whether this was associated with overt neuron loss, it does not seem implausible to speculate that the elevated levels of GCs in ischemic patients may exacerbate the E A A / N M D A / C a - ' + cascade and thus enhance neuronal loss. Although the clinical implication of this research awaits further investigation, it is conceivable that the findings of this study would expand our knowledge of ischemia pathophysiology and thus facilitate our ability to decrease ischemic brain damage.

Acknowledgements

We thank Sheila Brooke, Vinh Cao, Ingrid Chang, Michael Romero, Becky Stein, Jeremy Tompkins and Larry Tsai for expert technical assistance. Support was provided by N I H Grant AG06633 to R.M.S.

References

[1] Amoroso,D., El Tamer, A., Wulfert, E. and Hanin, I., Long-term exposure to corticosterone aggravates AF64A-induced cholinotoxicity in rat hippocampus, Soc. Neurosci. Abstr.. 19 (1993) 770.2. [2] Andine, P., Orwar, O., Jacobsom I., Sandberg, M. and Hagberg, H., Changes in extracellular amino acids and spontaneous neuronal activity during ischemia and extended reflow in the CAI of the rat hippocampus. J. Neurochem., 57 (1991) 222-229. [3] Armanini, M.P., Hutchins, C., Stein, B.A. and Sapolsky, R.M., Glucocorticoid endangerment of hippocampal neurons is NMDA-receptor dependent, Brain Res., 532 (1990) 7-12. [4] Balcar, V.J. and Johnston, G.A.R., The structural specificity of the high affinity uptake of t-glutamate and t,-aspartate by ral brain slices, J. Neurochem., 19 (1972) 2657-2666. [5] Beaudet, A., Burkhalter, A.. Reubi, J.-C. and Cuenod, M., Selective bidirectional transport of [-~H]D-aspartate in the pb geon retino-tectal pathway, Neuroscience, 6 (1981) 2021-2034. [6] Belhage, B., Rehder, V., Hansen, G.H., Kater, S.B. and Schousboe, A.. ~H-D-Aspartate release from cerebellar granule neurons is differentially regulated by glutamate- and K ~-stimulation, J. Neurosci, Res., 33 (1992)436-444. [7] Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J. Neurochem., 43 (1984) 1369-1374. [8] Cruickshank, J.M., Neil-Dwyer, G. and Stott, A.W., Possible role of catecholamines, corticosteroids and potassium in production of electrocardiographic abnormalities associated with subarachnoid hemorrhage, Br. Heart J., 36 (1974) 697-706. [9] Davies, L.P. and Johnston, G.A.R.. Uptake and release of D-

Y.-C. Chou et al./Brain Research 654 11994) 8-14 and L-aspartate by rat brain slices, J. Neurochem., 26 (1976) 1007-1014. [10] Drejer, J., Larsson, OM. and Schousboe, A., Characterization of L-glutamate uptake into and release from astrocytes and neurons cultured from different brain regions, Exp. Brain Res., 47 (1982) 2 59-269. [11] Drejer, J., Benveniste, H., Diemer, N.H. and Schousboe, A., Cellular origin of ischemia-induced glutamate release from brain tissue in viw) and in vitro, Z Neurochem., 45 (1985) 145-151. [12] Elliott, E,M. and Sapolsky, R.M., Corticosterone enhances kainic acid induced calcium elevation in cultured hippocampal neurons, J. Neurochem., 59 (1992) 1033-1040. [13] Elliott, E.M. and Sapolsky, R.M., Corticosterone impairs hippocampal neuronal calcium regulation - possible mediating mechanisms, Brain Res., 602 (1993) 84-90. [14] Elliott, E., Mattson, M., Vanderklish, P., Lynch, G., Chang, I. and Sapolsky, R., Corticosterone exacerbates kainate-induced alterations in hippocampal tau immunoreactivity and spectrin proteolysis in vivo, J. Neurochern., 61 (1993) 57-67. [15] Erecinska, M. and Silver, I.A., Metabolism and role of glutamate in mammalian brain, Prog. Neurobiol., 35 (1990) 245-296. [16] Feibel, J.H., Hardy, P.M., Campbell, R.G., Goldstein, M.N. and Joynt, R.J., Prognostic value of the stress response following stroke, J. Am. Med. Assoc., 238 (1974) 1374-1376. [17] Freo, U., Holloway, H.W., Kalogeras, K., Rapoport, S., Soncrant, T., Adrenalectomy or metyrapone-pretreatment abolishes cerebral metabolic responses to the serotonin agonist l-(2,5)-dimethosy-4-iodophenyl)2-aminopropane (DOI) in the hippocampus, Brain Res., 586 (1992) 256-264. [18] Globus, M.Y.T., Busto, R., Martinez, E., Valdes, I., Dietrich, W.D. and Ginsberg, M.D., Comparative effect of transient global ischemia on extracellular levels of glutamate, glycine, and gama-aminobutyric acid in vulnerable and non-vulnerable brain regions in the rat, 3. Neurochem., 57 (1991) 470-478. [19] Goldberg, M.P., Weiss, J.H., Pham, P.-C. and Choi, D.W., N-Methyl-D-Aspartate receptors mediate hypoxic neuronal injury in cortical cultures, J. Pharmacol. Exp. Ther., 243 (1987) 784-791. [20] Hagberg, H., Lehmann, A., Sandberg, M., Nystrom, B., Jacobson, 1. and Hamberger, A., Ischemia-induced shift of inhibitory and excitatory amino acids from intra- to extracellular compartments, J. Cereb. Blood Flow Metab., 5 (1985) 413-419. [21] Hall, E.D., Steroids and neuronal destruction or stabilization, Ciba Foundation Syrup., 153 (1990)206-219. [22] Horner, H.C., Packe, D.R. and Sapolsky, R.M., Glucocorticoids inhibit glucose transport in cultured hippocampal neurons and gila, Neuroendocrinology, 52 (1990) 57-64. [23] Hortnagl, H., Berger, M., Havelec, L. and Hornykiewicz, O,, Role of glucocorticoids in the cholinergic degeneration in rat hippocampus induced by ethylcholine aziridinium, J. Neurosci., 13 (1993) 2939-2945. [24] Johnson, M., Stone, D., Bush, L., Hanson, G. and Gibb, J., Glucocorticoids and 3,4-methylenedoixymethamphetamine-induced toxicity, Eur. J. PharmacoL, 161 (1989) 181 185. [25] Kadekaro, M., lto, M. and Gross, P,M., Local cerebral glucose utilization is increased in acutely adrenalectomized rats, Neuroendocrinology, 47 (1988) 329-334. [26] Kanai, Y., Smith, C.P. and Hediger, M.A., The elusive transporters with a high affinity for glutamate, Trends Neurosci., 16 (1993) 365-370. [27] Kauppienen, R., Enkvist, K., Holopainen, I. and Akerman, K., Glucose deprivation depolarizes plasma membrane of cultured astrocytes and collapses transmembrane potassium and glutamate gradients, Neuroscience, 26 11988) 283-290. [28] Koide, T., Wieloch, T. and Siesjo, B., Chronic dexamethasone pretreatment aggravates ischemic neuronal necrosis, J. Cereb, Blood Flow Metab., 6 (1986) 395-4/t6.

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[29] Lowy, M., Gault, L., Yamamoto, B., Adrenalectomy attenuates stress-induced elevations in extracellular glutamate concentrations in the hippocampus, J. Neurochem., 61 (1993) 1957-196lL [30] Maycox, P.R., Hell, J.W. and Jahn, R., Amino acid neurotransmission: spotlight on synaptic vesicles, Trends Neurosci., 13 (1990) 83-87. [31] McCaslin, P.P. and Yu, X.Z., Cyanide selectively augments kainate- but not NMDA-induced release of glutamate and tau rine, Eur. J. Pkarmacol., 228 11992) 73-75. [32] Moghaddam, B., Stress preferentially increases extraneuronal levels of excitatory amino acids in the prefrontal cortex: Comparison to hippocampus and basal ganglia, J. Ncurochem.. 60 (1993) 1650-1657. [33] Morse, J.K. and Davis, J.N., Regulation of ischemic hippocampal damage in the gerbil: adrenalectomy alters the rats of CA1 cell disappearance, Exp. Neurol., 110 (1990) 86 92. [34] Mulley, G.P., Wilcox, R.G. and Harrison. M.J.G.. Plasma cortisol as a m e a s u r e o f stress response in acute stroke, Stroke, 20 (1989) 1593. [35] Naito, S. and Ueda, T,, Characterization of glutamate uptake into synaptic vesicles, J. Neurochem., 44 (1985) 99 1{)9. [36] Nicholls, D.G., Release of glutamate, aspartate, and yaminobutyric acid from isolated nerve terminals. J. Neurochem., 52 (1989) 331 341. [37] Nicholls. D., and Attwell, D.. The release and uptake of excitatory amino acids, Trends Pharmacol. Sci., I1 11990) 462-468. [38] Olsson, T.. Urinary free cortisol excretion shortly after ischaemic stroke, J. htt. Med., 228 (1990) 177-18[. [39] Olsson, T., Astrom, M., Eriksson, S. and Forssell, A_ Hypercortisolism revealed by the dexamethasone suppression test with acute ischemic stroke, Stroke, 2(1 (1989) 1685-169l). [40] Olsson, T., Marklund, N., Gustafson. Y. and Nasman, 13., Abnormalities at different levels of the hypothalamic-pituitatTadrenocortical axis early after stroke. Stroke, 23 (1992) 1573 1576. [41] O'Neill, P.A., Davies, l., Fullerton, K.J. and Bennett, D., Stress hormone and blood glucose response following acute stroke in the elderly, Stroke, 22 11991 ) 842-847. [42] Palaiologos, G., Hertz, L. and Schousboe, R., Evidence that aspartate aminotransferase activity and ketodicarboxy[atc carrier function are essential for biosynthesis of transmitter glutamate, J. Neurochem., 51 (1988) 317 320. [43] Patel, M.N., Ardelt, B.K.. Yim, G.K.W. and lson, G.E., Cyanide induces Ca 2 +-dependent and -independent release of glutamate from mouse brain slices, Neurosci. Lett., 131 (1991) 42-44. [44] Patel, M.N., Yim, G.K.W. and lson. G.E., Blockade of Nmethyl-D-aspartate receptors prevents cyanide-induced neuronal injury in primary hippocampal cultures, Toaiuol. Appl. Pharmaco/.. 115 (1992) 124-129. [45] Rothman, S., Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death. J. Nettro~'ci., 4 (1984) 1884-1891. [46] Sanchez-Prieto, J., and Gonzalez. P., Occurrence of a large CaZ~-independent release of glutamate during anoxia in isolated nerve terminals (Snaptosomes). J. NeHrochem.. 5() (1988) 1322-1324. [47] Sapolsky, R.. A mechanism for glucocorticoid toxicity in the hippocampus: increased neuronal vulnerability to metabolic insuits, J. Neurosci.. 5 (1985) 1227-1231. [48] Sapolsky, R.M., Glucocorticoid toxicity in the hippocampus: reversal by supplementation with brain fuels, .L Neuro.wi., 6 (1986) 2240-2244. [49] Sapolsky, R.M., Glucocorticoids, hippocampal damage and the glutamatergic synapse, Prog. Brain Res., 86 11990) 13 23. [50] Sapolsky. R.M. and Pulsinelli, W.A., Glucocorticoids potentiate ischemic injury to neurons: therapeutic implications. Science, 229 (1985) 1397-1399.

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[51] Sarantis, M., Ballerini, L., Miller, B., Silver, R.A., Edwards, M. and Attwell, D., Glutamate uptake from the synaptic cleft does not shape the decay of the non-NMDA component of the synaptic current, Neuron, 11 (1993) 541-549. [52] Stein-Behrens, B.A., Elliott, E.M., Miller, C.A., Schilling, J.M., Newcombe, R. and Sapolsky, R.M., Glucocorticoids exacerbation kainic acid-induced extracellular accumulation of excitatory amino acids in the rat hippocampus, J. Neurochem., 58 (1992) 173~) 1735. [53] Szerb, J. and O'Regan, P., Increase in the stimulation-induced overflow of excitatory amino acids from hippocampal slices: interaction between low glucose concentrations and fluoroacetate, Neurosci. Lett., 86 (1988) 207--211. [54] Theoret, Y., Caldwell-Kenke[, J. and Krigman, M., The role of neuronal metabolic insult in organometal neurotoxicity, Toxicologist, 6 (1985) 491.

[55] Tombaugh, G.C., Yang, S.tt., Swanson, R.A. and R.M. Sapolsky, Glucocorticoids exacerbate hypoxic and hypoglycemic hippocampal injury in vitro: biochemical correlates and a role Rw astrocytes, Z Neurochem., 59 (1992) t37-146, [56] Virgin Jr., C.E., Ha, T.P.-T., Packan, D.R. Tombaugh, G.C., Yang, S.tt., Horner, H.C. and Sapolsky, R.M., Gh.cocorticoids inhibit glucose transport and glutamate uptake in hippocampal astrocytes: implications for glucocorticoid neurotoxicity, ,I. Neurochem., 57 (1991) 1422-1428. [57] Yu, A.C.H., Hertz, E. and Hertz, L., Alterations in uptake and release rates for GABA, glutamate, and glutamine during biochemical maturation of highly purified cultures of cerebral cortical neurons, a GABAergic preparation, J. Neurochem., 42 (1984) 951-960.