Plasticity of Excitatory Amino Acid Transporters in Experimental Epilepsy

Epilepsiu,41(Suppl. 6):s104-51 10, 2000 Lippincott Williams & Wilkina, Inc., Baltimore 0 International League Against Epilcpsy

Plasticity of Excitatory Amino Acid Transporters in Experimental Epilepsy "0.I. Claudio, "f. Ferchmin, $L. VeliSek, $E. F. Sperber, $S. L. Moshk, and *J. G. Ortiz *Department of Pharmacology und Toxicology, University of Puerto Rico, School of Medicine, Sun Juan, Puerto Rico, U.S.A.; fDepartment cf Biochemistry, Universidad Central del Caribe, Bayamdn, Puerto Rico, U.S.A.; and $Department of Neurology, Albert Einstein College of Medicine, Bronx, New York, U.S.A.

Summary: Purpose: To examine the relationship between seizures and excitatory amino acid transporter (EAAT) activity and whether up-regulation of EAAT activity alters epileptogenicity. Methods: In this study, we exposed rat hippocampal slices to different convulsants before measuring EAAT activity. Rats were exposed to the EAAT inhibitor pyrrolidine-2,4dicarboxylic acid (PDC) before entorhinal cortex/hippocampal slices were obtained. These slices were exposed to low-Mg2+ buffer while electrophysiological recordings were obtained from the entorhinal cortex. mGluR I11 acting agents were used to study whether activation of mGluR I11 could regulate EAAT activity and if this regulation could overcome the effects on EAAT activity induced by the convulsants. Results: Veratridine, kainic acid (KA), and pilocarpine reduced EAAT activity in rat hippocampal slices. ~-2-Amino-4phosphonobutyric acid (an mGluR I11 agonist) restored EAAT activity and reduced epileptiform activity to near control levels.

The saturation curve for glutamate uptake in slices from KAseized rats killed 2 hours after the first forelimb clonus was displaced to the left, suggesting a compensatory change for the enhanced excitation. On the other hand, rats injected with the EAAT inhibitor PDC (by intracerebroventricular injection) had more severe KA-induced seizures and N-methyl-D-aspartate epileptiform activity than control rats. Furthermore, hippocampal slices from KA- or KA+PDC-treated rats exposed to low Mg2+reduced their firing rate to nearly zero once they returned to normal solution, whereas their control counterparts continued to fire, although at a lower rate. Conclusions: These results suggest a significant contribution of EAATs in some experimental epilepsy models and point to their short-term regulation by mGluR 111 as a possible source of their plasticity. Key Words: EAAT-Glutamate-UptakeSeizures-~-AP4.

The role of EAATs in epilepsy remains controversial

Excessive activation of excitatory amino acid [such as glutamic acid (GLU)] receptors is believed to result in pathological increases in Ca2+ concentration and oxidative stress and, ultimately, in massive neuronal loss (14). Binding to and subsequent transport of GLU by specialized excitatory amino acid transporters (EAATs 1 to 5) keep the extracellular GLU levels in the low range (1 to 5 pmol/L) (5-8). Arachidonic acid, nitric oxide, protein kinase C, and redox sites have been shown to alter EAAT activity (9). Excitotoxicity attributable to EAAT dysregulation has been implicated in many neuropathological conditions, including amyotrophic lateral sclerosis, ischemic insult, epilepsy, Parkinson's disease, Alzheimer's disease, and acquired immunodeficiency syndrome (10-14).

(15). No changes in astrocytic glutamate transporter (EAAT-2) or rat glutamate/aspartate transporter (EAAT1) are observed in kindled animals (16,17). On the other hand, mice deficient in astrocytic glutamate transporter manifest spontaneous seizures, and EAAT-3 expression is enhanced in rats with stage 5 kindling (1 1,16-18). Contrary to the expected decrease in EAAT activity, we observed that mice that were genetically susceptible to audiogenic seizures had increased GLU uptake (1 9). In humans, Tessler et al. (20) reported no difference in EAAT by Western blotting in patients with temporal lobe epilepsy. However, Mathern et al. (21) found increased EAAT-3 in the remaining granular and pyramidal cells, decreased EAAT-2 in the hilus and CAI stratum radiatum, and increased EAAT-1 in CA2 and CA3 of patients with temporal lobe epilepsy. This report examines the relationship between seizures and EAAT activity and whether up-regulation Of EAAT activity alters epileptogenicity.

Address correspondence and reprint requests to Dr. J. G. Ortiz at Department of Pharmacology and Toxicology, University of Puerto Rico School of Medicine. P.O. Box 365067. San Juan. Puerto Rico 00936-5067, U.S.A. E-mail [email protected]

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METHODS Animals Female Sprague-Dawley rats (125 to 150 g, 2 to 4 months old) were obtained from the University of Puerto Rico, Rio Piedras campus, or from the Universidad Central del Caribe School of Medicine. All animals had free access to food and water and were kept on a 12-hour light/dark cycle. Chemicals ~-[2,3,4-~H]Glutamic acid (60 Cdmmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO, U.S.A.). ~(+)-2-Amino-4-phosphonobutyricacid (LAP4) and (R,S)-methylserine-O-phosphate (MSOP) were obtained from Tocris Cookson (Ballwin, MO, U.S.A.). Ecolume was obtained from ICN Biomedicals (Irvine, CA, U.S.A.). All other reagents were obtained from Sigma Chemical (St. Louis, MO, U.S.A.) and were of the highest purity available. Hippocampal [3H]glutamateuptake Rats were lightly anesthetized with ether and decapitated. Hippocampi were promptly removed, sliced using a McIlwain tissue slicer, and kept in cold oxygenated (95% 0,/5% CO,) artificial cerebrospinal fluid (ACSF; 1.8 mmol/L CaCl,, 116.36 mmoln NaC1, 5.36 mmol/L KCl, 10 mmol/L D-glucose, 0.81 mmol/L MgSO,, 0.88 mmol/L Na,HPO,, 25 mmol/L NaHCO,). The slices were incubated for 1 hour at 37°C with ACSF with the different agents (i.e., L - A P ~diluted in ACSF) in a humidified atmosphere saturated with 95% 0,/5% CO,. Samples incubated at 60°C served as controls. The medium was promptly removed and replaced with ACSF containing labeled glutamate and further incubated for 10 min at 37°C. The reaction was stopped by aspiration of solutions. Slices were washed three times with 100 pL of cold ACSF. The slices were solubilized in 50 pL of 0.5 mol/L NaOH overnight. The radioactivity was quantified after acidification with 50 pL of glacial acetic acid and addition of 1 mL of Ecolume using a Beckman (Fullerton, CA, U.S.A.) LS 1800 scintillation counter. Electrophysiology Rats were decapitated and the brain promptly removed. The hippocampi were dissected on ice while being irrigated with ice-cold ACSF. A manual slicer or a vibratome was used to obtain transverse 400-km hippocampal slices. The slices were quickly transferred to an interface recording chamber and kept on a nylon mesh at the interface of humidified 95% 0,/5% CO, and ACSF at 35°C. The slices were allowed to stabilize undisturbed for 1 hour before recording. Concentric bipolar electrodes delivered stimulation. Constant current stimuli were generated by an S48 stimulator and a PSIU6 stimulus isolation unit (Grass). Micropipets filled with 2 m o l n NaCl and impedance ranging from 1 to 5 M a functioned

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as recording electrodes. The recording electrodes were placed in the stratum radiatum, stratum pyramidale, enthorrinal cortex, or a combination of these sites. Pair stimuli of 0.2-millisecond duration and 20-millisecond interpulse intervals were delivered in the stratum radiatum every 60 seconds when recording from the hippocampus. The average strength of the stimuli was set to obtain 50 to 60% of the maximal response. Unless stated otherwise, values ranged from 50 to 170 FA. The population responses were amplified (P511 Grass amplifier) and recorded in a chart recorder or digitized and stored for further analysis with the LABMAN system.

Protein determination The protein concentration was measured as described by Bradford (22) using bovine serum albumin as a standard. Statistical analysis The statistical analysis (Student t test, analysis of variance) was performed using Instat version 2.2 obtained from GraphPad (San Diego, CA, U.S.A.). RESULTS Fig. I shows the effects of different convulsants on EAAT activity in rat hippocampal slices. Veratridine (1 mmol/L) and kainic acid (KA) caused a marked decrease in EAAT activity. In contrast, depolarization by 50 mmol/L K+, pilocarpine, or 4-aminopyridine or zero Mg2+ [activation of N-methyl-D-aspartate (NMDA) receptors] did not affect hippocampal EAAT activity measured at 50 pmolL. The inhibitory effects of veratridine on hippocampal EAAT activity were partially reversed by coincubation with the mGluR I11 agonist L - A P (1 ~ mmol/L; Fig. 2A). The mGluR I11 antagonist MSOP had no effect on EAAT activity by itself. Furthermore, it did not reverse the veratridine-induced reduction of EAAT activity (Fig. 2A, inset). The epileptiform activity induced by veratridine in hippocampal slices (Fig. 2B) was reversed almost immediately by superfusion with L-AP4. In contrast, veratridine-exposed slices required at least 120 minutes of superfusion with ACSF to show signs of recovery. KA-injected rats (10 mgkg intraperitoneal) killed 2 hours after the first forelimb clonus exhibited a marked shift to the left in their EAAT activity curve (Fig. 3). This effect was not observed if the rats were killed 2 days later (Fig. 3, inset). Thus, different stages of the same convulsant had markedly different effects on EAAT activity. Fig. 4 shows that entoshinal cortexkippocampal slices exhibited an increase in the number of spikes when exposed to ACSF without Mg2+ (activation of NMDA receptors), as expected. Once normal Mg2+concentrations were reestablished, the number of spikes was reduced by approximately 50%. Slices from rats injected with KA and killed 2 hours later exhibited a pattern similar to that of the saline-injected rats described above, except that Epilepsia, Vol. 41, Suppl. 6, 2000

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FIG. 1. Effects of different convulsants on EAAT activity in rat hippocampal slices. After incubating the slices for 1 hour with the convulsant, EAAT activity was measured by incubating the slices for 10 minutes with 50 VmollL [3H]glutamate. Slices incubated at 60°C for 1 hour and treated similarly served as controls. The incubation was terminated by aspirating the [3H]glutamate solution and washing the slices three times with ACSF. PTZ, pentyletetrazole (5 mmol/L); KA, kainic acid (5 mmol/L); PILO, pilocarpine (5 mmollL); 4AP, 4-aminopyridine (5 mmollL); VERA, veratridine (1 mmol/L). **p < 0.05 versus 37°C; *** p < 0.001 versus 37°C.

reestablishment of normal Mg2+ concentrations resulted in total cessation of the spikes. In contrast, slices from rats pretreated with the EAAT inhibitor pyrrolidine-2,4dicarboxylic acid (PDC; intracerebroventricular injection) and injected with KA fired at roughly twice the rate as those from control or KA-injected rats alone. Once normal Mg2+concentrations were reestablished, their firing essentially stopped similarly to what was observed in KA-injected rats.

DISCUSSION High-frequency activation of hippocampal slices causes a decrease in EAAT activity (23,24). Our results show that some convulsants, such as veratridine, are capable of reducing EAAT activity, whereas others (e.g., 50 mmol/L K+) have no detectable effect independent of having equivalent mechanisms of action and causing similar epileptiform activity. Epilepsia, Vol. 41, Suppl. 6, 2000

It is generally agreed that the energetic loss that accompanies seizures and ischemia results in cessation of Na+-K+ATPase activity (25). In turn, Na+ accumulates inside the presynaptic terminal, causing the reversal of glutamate transporters (transporter reversal hypothesis) and hence the increased extracellular glutamate observed in many pathological conditions (26,27). The fact that different convulsants have differential effects on EAAT activity in hippocampal slices raises questions about this hypothesis. Furthermore, most of the glutamate transport is performed by glia [rat glutamate/aspartate transporter and astrocytic glutamate transporter (28-30)], and EAAT reversal does not occur at extracellular potassium concentrations attainable in brain (31,32). The anticonvulsant properties of mGluR 111 agonists have been reported (33-36). Accordingly, our results demonstrate that EAAT activity can be up-regulated by L - A P ~[similar observations were obtained with the mGluR I/II antagonist MCPG (data not shown)] and that

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FIG. 2. A: Veratridine (VERA; 1 mmol/L) causes a marked decrease in EAAT activity in rat hippocampal slices. L - A P by ~ itself has no effect, although it is capable of partially reversing the inhibitory effect of veratridine on EAAT activity. The insert shows that the mGluR 111 antagonist MSOP has no effect on EAAT activity or reverses the veratridine-induced reduction of EAAT activity. **p < 0.01, ***p < 0.001 111 versus 37°C;+++p < 0.001 versus L - A P ~ + MSOP (inset); ###p < 0.001 versus veratridine. B: L - A P ~is also capable of reducing the veratridine-induced epileptiform activity in the hippocampal slices almost immediately, whereas superfusion of similarly treated slices requires more than 2 hours to recover.

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FIG. 3. The EAAT activity in hippocampal slices from rats treated with KA (10 mglkg intraperitoneal) and killed 2 hours after the first forelimb clonus is displaced to the left compared with the EAAT activity in slices of saline-injected rats (CTL). The inset show that the EAAT activity in slices of KA-treated rats that did not reach status epilepticus (no damage) and allowed to survive for 48 hours is not different from that obtained from saline-injected rats.

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FIG. 4. Effects of NMDA activation caused by removal of Mg2+in hippocampal slices from rats injected with saline (intracerebroventricular)/saline (intraperitoneal) (sahal), saline (intracerebroventricular)/KA(intraperitoneal) (sal/ka), or PDC (intracerebroventricular)/KA (pdc/ka). Slices from salinehaline- or saline/KA-treated rats have similar increases in their firing rate during the transition from normal ACSF to no-Mg"' ACSF (Mg" out) and while the no-Mg2+ACSF (no Mg+') is being superfused. In contrast, slices from PDC/KA-treated rats fire approximately twice as much as those from salinetsaline- or saline/KA-injected rats during the no-Mg2+ ACSF superfusion (no Mg+'). During the return to normal Mg2+ACSF (Mg'" in), both saline/KA- and PDC/KA-treated slices continue to fire, whereas a significant reduction is observed in the saline/saline-treated slices. However, upon returning to normal ACSF (WASH), slices of PDC/KA- and saline/KA-treated rats greatly diminish their firing rate, whereas salinekaline-treated slices continue to fire. ***p < 0.001 versus saline/ saline; +++p < 0.001 versus saline/KA.

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PLASTICITY OF EAAT AND SEIZURES superfusion to veratridine-treated hippocampal slices with L - A P ~causes almost immediate cessation of the epileptiform activity. These observations point to the therapeutic potential of mGluR I11 agonists. In hippocampal slice cultures, inhibition of EAAT-3 expression by antisense knockdown shows differential KA toxicity of CA3 and CA1 (37). Yet, in intact rats, a decrease in EAAT-3 immunoreactivity is observed in the stratum lacunosum moleculare 4 hours after the onset of seizures. Upon pyramidal cell loss (5 days after seizures), a total loss of EAAT-3 immunoreactivity is observed in CA1 and the stratum lacunosum moleculare (38). Our experiments show a rapid up-regulation of EAAT activity in hippocampal slices of rats killed 2 hours after the first forelimb clonus but not allowed to go into status epilepticus, whereas similarly treated rats allowed to survive for 48 hours (damage requires status epilepticus) have essentially the same EAAT activity as saline-injected rats. Together, these observations indicate that a given convulsant can have different effects on EAAT at different times. It is not surprising that hippocampal slices from rats treated with the glutamate uptake inhibitor PDC (by intracerebroventricular injection) and with KA have higher firing rates when exposed to no Mg2+ (activation of NMDA receptors) than those treated with saline/KA or saline/saline alone even though they were superfused with ACSF for 1 hour. Interestingly, slices from saline/ saline-injected rats do not decrease their firing rate to zero once the Mg2+ concentration is returned to normal. These results are consistent with the potentiation by KA (GluR V) of y-aminobutyric acid-mediated inhibition observed by Cossart et al. (39). Our results clearly show the following: (a) different convulsants have different effects on EAAT activity; (b) L - A P ~reverses veratridine-induced decreases in EAAT activity and veratridine-induced epileptiform activity; (c) EAAT activity changes vary depending on the stage of the convulsant being examined; and (d) inhibition of EAAT activity results in higher firing rates. In addition to indicating the therapeutic potential of up-regulating EAAT activity, these results also may be useful in understanding the current literature on EAAT and seizures. Acknowledgment: This work was supported in part by the Minority Biomedical Research Support Program (University of Puerto Rico) and by the Research Centers for Minority Institutions (University of Puerto Rico and Universidad Central del Caribe). The support of the Animal Resources Centers at the Universidad Central del Caribe (B. Torres) and the University of Puerto Rico Rfo Piedras campus (L. Rosario) is hereby acknowledged.

REFERENCES 1 Carried0 SG, Yin HZ, Sensi SL, Weiss JH. Rapid Ca2+ entry through Ca2+-permeableAMPMainate channels triggers marked intracellular Ca2' rises and consequent oxygen radical production. J Neurosci 1998;18:7727-38,

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2. Urushitani M, Shimohama S, Kihara T, et al. Mechanism of selective motor neuronal death after exposure of spinal cord to glutamate: involvement of glutamate-induced nitric oxide in motor neuron toxicity and nonmotor neuron protection. Ann Neurol 1998; 44:796-807. 3 Blanc EM, Keller J N , Fernandez S , Mattson M P . 4Hydroxynonenal, a lipid peroxidation product, impairs glutamate transport in cortical astrocytes. Glia 1998;22: 149-60. 4. Meldrum B, Garthwaite J. Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol Sci 1990;11:37987. 5. Kanai Y, Hediger MA. Primary structure and functional characterization of a high-affinity glutanate transporter. Nature 1992; 360467-9. 6. Hediger MA. Structure, function and evolution of solute transporters in prokaryotes and eukaryotes. J Exp Biol 1994;196:1549. 7. Lehre KP, Danbolt NC. The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain. J Neurosci 1998;lS: 8751-7. 8. Robinson MB. The family of the sodium-dependent glutamate transporters: a focus on the GLT- 1/EAAT2 subtype. Neurochem Int 1999;33:479-91. 9. Trotti D, Rizzini BL, Rossi D, et al. Neuronal and glial glutamate transporters possess an SH-based redox regulatory mechanism. Eur J Neurosci 1997;9:123643. 10. Rothstein JD, Jin L, Dykes-Hoberg M, Kuncl RW. Chronic inhibition of glutamate uptake produces a model of slow neurotoxicity. Proc Nut1 Acad Sci USA 1993;90:6591-5. 11. Rothstein JD, Dykes-Hoberg M, Pardo CA, et al. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 1996;16:67586. 12. Rao R, Baskaya VL, Dogan A, et al. Traumatic brain injury downregulates glial glutamate transporter (GLT-1 and GLAST) proteins in rat brain. J Neurochem 1998;70:2020-7. 13. Hazel1 AS, Itzhak Y, Liu H, Norenberg MD. 1-Methyl-4-phenylI ,2,3,6-tetrahydropyridine(MPTP) decreases glutamate uptake in cultured astrocytes. J Neurochem 1997;68:2216-9. 14. Vesce S , Bezzi P, Rossi D, et al. HIV-1 gp120 glycoprotein affects the astrocyte control of extracellular glutamate by both inhibiting the uptake and stimulating the release of the amino acid. FEBS Lett 1997;411: 107-9. 15. Meldrum BS, Abkar MT, Chapman AG. Glutamate receptors and transporters in genetic and acquired epilepsy. Epilepsy Res 1999; 36: 189-204. 16. Miller HP, Levey AL, Rothstein JD, et al. Alteration in glutamate transporter protein levels in kindling-induced epilepsy. J Neurochem 1997;68:1564-70. 17. Abkar MT, Torp R, Danbolt NC, et al. Expression of the glial transporters GLT-1 and GLAST is unchanged in the hippocampus in fully kindled rats. Neuroscience 1997;78:351-9. 18. Tanaka K, Watase K, Manabe T, et al. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 1997;276: 1699-702. 19. Cordero ML, Ortiz JG, Santiago G. High affinity [3H]glutamate uptake systems in normal and audiogenic seizure-susceptible mice. Dev Brain Res 1994;78:44-8. 20. Tessler S , Danbolt NC, Faull RL, et al. Expression of the glutamate transporters in human temporal lobe epilepsy. Neuroscience 1999; 88:1083-91. 21. Mathern GW, Mendoza D, Lozada A, et al. Hippocampal GABA and glutamate transporter immunoreactivity in patients with temporal lobe epilepsy. Neurology 1999;52:453-72. 22. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. 23. Wieraszko A. Uptake of D-[3H]aspartic acid by hippocampal slices: the influence of low and high frequency activation of nerve endings. Brain Res 1983;259:324-6. 24. Goh JW, Ho-Asjoe M, Sastry BR. Tetanic stimulation-induced changes in [3H]glutamate binding and uptake in rat hippocampus. Gen Pharmacol 1986;17:53742.

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25. Madl JE, Burgesser K. Adenosine triphosphate depletion reverses sodium-dependent, neuronal uptake of glutamate in rat hippocampal slices. J Neurosci 1993;13:4429-44. 26. Szakowski M, Attwell D. Triggering and execution of neuronal death in brain ischemia: two phases of glutamate release by different mechanisms. Trends Neurosci 1994;17:359-65. 27. Katsumori K, Baldwin RA, Wasterlain CG. Reverse transport of glutamate during depolarization in immature hippocampal slices. Brain Res 1999;819160-4. 28. Lehre KP, Danbolt NC. The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain. J Neurosci 1998;18: 8751-7. 29. Haugeto 0, Ullensvang K, Levy LM, et al. Brain glutamate transporter proteins form homomultimers. J Biol Chem 1996;271: 27715-22. 30. Bergles DE, Jahr CE. Glial contribution to glutamate uptake at Schaeffer collateral-commisural synapses in the hippocampus. J Neurosci 1998;18:7709-16. 31. Longueinare MC, Swanson RA. Net glutamate release from astrocytes is not induced by extracellular potassium concentrations attainable in brain. J Neurochem 1997;69:14 32. Duan S, Anderson CM, Stein BA, Swanson RA. Glutamate induces rapid upregulation of astrocyte glutamate transport and cellsurface expression of GLAST. J Neurosci 1999;19: 10193-200.

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33. Abdul-Ghani A-S, Attwell PJE, Kent NS, et al. Anti-epileptogenic and anticonvulsant activity of ~-2-amino-4-phosphonobutyrate, a presynaptic glutamate receptor agonist. Brain Res 1997;755:20212. 34. Suzuki K, Mori N, Kittaka H, et al. Anticonvulsant action of metabotropic glutamate receptor agonists in kindled amygdala of rats. Neurosci Lett 1996;204:41-4. 35. Ghauri M, Chapman AG, Meldrum BS. Convulsant and anticonvulsant actions of agonist and antagonists of group I11 mGluRs. NeuroReport 1996;7: 1469-74. 36. Gasparin F, Bruno V, Battaglia G, et al. (R,S)-4-Phosphonophenyglycine, a potent and selective group 111metabotropic glutamate receptor agonist, is anticonvulsive and neuroprotective in vivo. J Pharm.uco1 Exp Ther 1999;289:1678-87. 37. Simantov R, Liu W, Broutman G, Baudry M. Antisense knockdown of glutamate transporters alters subfiled selectivity of kainate-induced cell death in rat hippocampal slice cultures. J Neurochem 1999;73:1828-35. 38. Simantov R, Crispino M, Hoe W, et al. Changes in expression of neuronal and glial glutamate transporters in rat hippocampus following kainate-induced seizure activity. Mol Bruin Res 1999;65: 112-23. 39. Cossart R, Esclapez M, Hirsch JC, et al. GluR5 kainate receptor activation in interneurons increases tonic inhibition of pyramidal cells. Nut Neurosci 1998; 1:470-8.