Ampa receptor mediated excitotoxicity in neocortical neurons is developmentally regulated and dependent upon receptor desensitization

NEUROCHEMISTRY International Neurochem[ Int[ 21 "0887# 494Ð402

AMPA receptor mediated excitotoxicity in neocortical neurons is developmentally regulated and dependent upon receptor desensitization J[ B[ Jensen\ A[ Schousboe\ D[ S[ Pickering PharmaBiotec Research Center\ Department of Pharmacolo`y\ The Royal Danish School of Pharmacy\ DK!1099 Copenha`en\ Denmark Received 19 October 0886^ accepted 0 November 0886

Abstract AMPA "a!amino!2!hydroxy!4!methyl!3!isoxazolepropionic acid# excitotoxicity was examined in cultured neocortical neurons using the reduction of 2!"3\4!dimethylthiazol!1!yl#!1\4!diphenyltetrazolium bromide "MTT# to measure cell viability[ Neurons were exposed to AMPA at di}erent culture periods during development of the neurons[ In order to describe the pharmacology of AMPA! mediated toxicity\ several glutamate receptor antagonists were used] MK!790\ NS 283\ NBQX\ GYKI 41355\ GYKI 42394 and GYKI 42544[ Increased excitotoxicity was observed when cortical neurons cultured for 4\ 7 and 01 days in vitro "DIV# were exposed to a high concentration of AMPA "499 mM# for 5 h[ However\ only at DIV 01 was part of the toxicity mediated directly through AMPA receptors since 09 mM MK!790 blocked all AMPA toxicity at DIV 4 and 7\ but only some of the AMPA response at DIV 01[ This indicated that NMDA receptors were being activated\ causing some of the observed toxicity[ The high dose of AMPA was not su.cient to damage all neurons since 48) remained viable after exposure to AMPA even for neurons that were cultured for 01 DIV[ Since it is known that both glutamate and AMPA activate AMPA receptors with a fast and rapidly desensitizing response\ this could explain the relatively low toxicity produced by 499 mM AMPA[ This was investigated by blocking AMPA receptor desensitization with cyclothiazide[ Using a lower concentration "14 mM# of AMPA\ addition of 49 mM cyclothiazide increased the AMPA induced excitotoxicity in cultured cortical neurons at all DIV except for DIV 1[ This combination of AMPA¦cyclothiazide yielded 66) cell death for DIV 01 cultures[ In contrast to the results observed with 499 mM AMPA\ the neurotoxicity mediated directly by AMPA receptors when desensitization was blocked was seen as early as 4 DIV since 09 mM MK! 790 did not completely block the response whereas 09 mM NBQX did[ The 1\2!benzodiazepine GYKI compounds\ which have been reported to be selective non!competitive AMPA receptor antagonists\ were here observed to block the AMPA toxicity with the following rank order] GYKI 42544 × GYKI 41355 − GYKI 42394\ which is in agreement with their published potencies[ Þ 0887 Elsevier Science Ltd[ All rights reserved[

0[ Introduction Glutamate\ the major excitatory amino acid in the brain\ can induce neurotoxic e}ects by overstimulation of glu! tamate receptors "Olney\ 0877#[ The excitotoxicity seen in neurons after excessive glutamate stimulation is thought to be mediated through the ionotropic glutamate receptors "Choi\ 0881#[ Brief exposure of either glutamate or NMDA leads to rapidly induced neuronal cell death due to activation of NMDA receptors "Kim et al[\ 0876^ Choi et al[\ 0877#[ In contrast\ prolonged activation of the non!NMDA receptors is necessary to mediate exci! totoxicity when neurons are stimulated by AMPA or kainate "Koh et al[\ 0889#[ It has been proposed that  To whom all correspondence should be addressed ðFax] "¦34# 24 26 95 22Ł[ 9086Ð9075:87 ,08[99 Þ 0887 Elsevier Science Ltd[ All rights reserved PII] S 9 0 8 6 Ð 9 0 7 5 " 8 6 # 9 9 0 2 9 Ð 6

calcium in~ux and subsequent elevation of intracellular free calcium concentration "ðCa1¦Łi# is crucial in triggering neuronal death after exposure to excitatory amino acid "EAA# agonists "Choi\ 0874#[ The rapidly triggered exci! totoxicity seen upon brief stimulation with glutamate or NMDA is re~ected by the highly calcium!permeable NMDA receptor channels\ which consequently leads to high calcium in~ux and rise in ðCa1¦Łi prior to exci! totoxicity "Choi\ 0881#[ AMPA\ which has previously been shown to mediate cell death in cultured neocortical neurons "Frandsen and Schousboe\ 0876^ Koh et al[\ 0889#\ was initially thought to mediate injury by stimu! lation of calcium in~ux through voltage!gated Ca1¦ chan! nels since most AMPA channels have limited calcium permeability and since calcium channel blockers could reduce AMPA mediated toxicity "Weiss et al[\ 0889#[ However\ since K¦!stimulated Ca1¦ in~ux is non!toxic

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to neurons "Frandsen and Schousboe\ 0880#\ the role of voltage!gated calcium channels in AMPA mediated toxicity is unclear[ Recently it was shown\ that long!term exposure to AMPA can induce substantial neuronal death involving apoptosis in cultured cortical neurons "Larm et al[\ 0886#[ However\ in some studies AMPA has been shown to produce little excitotoxicity in neuronal cultures even at prolonged exposure times and that inhibition of AMPA receptor desensitization is necessary to provoke toxic events "May and Robison\ 0882#[ The activation of non! NMDA receptors by glutamate and AMPA has been shown to produce a fast and rapidly desensitizing response in neurons "Kiskin et al[\ 0875^ Patneau and Mayer\ 0880#[ The desensitizing e}ect on non!NMDA receptors can be blocked by plant lectins "Thio et al[\ 0881#\ the nootropic drug aniracetam "Tang et al[\ 0880# and benzothiadiazides\ including cyclothiazide "Yamada and Tang\ 0882^ Moudy et al[\ 0883#[ By blocking the desensitizing e}ect of AMPA\ 34Ca1¦ uptake\ intra! cellular calcium concentration "Cebers and Liljequist\ 0884^ Hoyt et al[\ 0884# and cell death "May and Robison\ 0882^ Moudy et al[\ 0883#\ have been shown to increase upon AMPA receptor stimulation[ Functional studies of the AMPA receptors have shown that the subunit composition of the receptor complex is very important for determining the kinetic properties of the receptor including desensitization\ deactivation and recovery rate "Hollmann et al[\ 0880^ Burnashev\ 0882^ Swanson et al[\ 0886^ Angulo et al[\ 0886#[ It has been shown that desensitization can be regulated by the di}er! ent ~ip and ~op versions of the receptor subunits "Mosbacher et al[\ 0883^ Lambolez et al[\ 0885^ Partin et al[\ 0885# whereas recovery from desensitization can be controlled by the R:G editing site of either GluR1\ GluR2 or GluR3 "Lomelie et al[\ 0883#[ The exact subunit com! position of the channel is also important for determining the desensitization properties of the receptor complex[ For example\ GluR3 has been shown to dominate the desensitization response when co!expressed with either GluR1 ~ip or ~op versions "Mosbacher et al[\ 0883^ Geiger et al[\ 0884#[ Also\ the calcium permeability of the receptor channel is critically determined by the receptor subunit composition\ the presence of GluR1"R# subunit giving receptors with low calcium permeability "Hollmann et al[\ 0880#[ These _ndings clearly demonstrate that di}erent neu! rons contain AMPA receptors with diverse functions[ Depending on the nature of the AMPA receptor complex\ AMPA mediated excitotoxicity in vivo could play an important role during stroke\ ischemia and other EAA! induced injuries[ As an initial examination of AMPA receptor mediated toxicity in cultured neocortical neu! rons it was decided to investigate whether the develop! mental pro_le of AMPA excitotoxicity was dependent upon receptor desensitization[ Receptor desensitization

was blocked by treatment of the neurons with cyclo! thiazide "Yamada and Tang\ 0882# and various EAA receptor antagonists were used in order to phar! macologically characterize the AMPA mediated exci! totoxicity[ The non!competitive NMDA receptor antagonist MK!790 "Wong et al[\ 0875# was used to block responses mediated by NMDA receptors and the com! petitive antagonists NBQX "Sheardown et al[\ 0889# and NS 283 to block non!NMDA receptors[ The compound NS 283 inhibits both AMPA and kainate receptors in cultured hippocampal neurons with IC49 values of 9[34 mM and 9[02 mM respectively "Paternain et al[\ 0885#[ The 1\2!benzodiazepine GYKI compounds\ which have previously been shown to be non!competitive non! NMDA receptor antagonists "Donevan and Rogawski\ 0882^ Vizi et al[\ 0885#\ were also used as AMPA receptor antagonists[ It was found that excitotoxicity mediated directly through AMPA receptors is highly dependent upon receptor desensitization and the toxicity produced increases during development of the cultures[

1[ Experimental procedures 1[0[ Materials Pregnant mice "NMRI# were obtained from the animal quarters at the Panum Institute "University of Copen! hagen\ Denmark# and Bomholt Farm "Ry\ Denmark#[ Plastic tissue culture plates "13!wells# were purchased from NUNC A:S "Denmark#[ Fetal calf serum was obtained from Sera!Lab "Sussex\ U[K[#\ insulin from NOVO Nordisk "Denmark# and penicillin from Leo "Denmark#[ Cytosine b!D!arabinofuranoside\ trypsin\ soybean trypsin inhibitor\ DNAse\ L!glutamine\ p! aminobenzoic acid\ poly!D!lysine "mol wt × 299\999#\ sodium dodecyl sulfate "SDS# and 2!"3\4!dimethyl! thiazol!1!yl#!1\4!diphenyltetrazolium bromide "MTT# were obtained from Sigma "St[ Louis\ MO\ U[S[A[#[ "RS#!a!amino!2!hydroxy!4!methyl!3!isoxazolepropionic acid "AMPA# was synthesized in the laboratory of Dr P[ Krogsgaard!Larsen "Department of Medicinal Chem! istry\ The Royal Danish School of Pharmacy\ Copen! hagen\ Denmark#[ GYKI compounds\ 0!"3!amino! phenyl#!3!methyl!6\7!methylenedioxy!4H!1\2!benzodia! zepine "GYKI 41355#\ 0!"3!aminophenyl#!2!acetyl!3! methyl!2\3!dihydro!6\7!methylenedioxy!4H!1\2!benzo! diazepine "GYKI 42394# and 0!"3!amino!phenyl#!2! methylcarbamyl!3!methyl!2\3!dihydro!6\7!methylene! dioxy!4H!1\2!benzodiazepine "GYKI 42544# were a gen! erous gift from Dr Pal Somogyi "Institute for Drug Research\ Ltd[\ Hungary#[ 7!Methyl!4!"3!nitrophenyl#! 5\6\7\8!tetrahydro!0H!pyrroloð2\1!hŁisoquinoline!1\2! dione!2!oxime "NS 283# was kindly provided by Dr J[ Drejer "NeuroSearch A:S\ Glostrup\ Denmark#[ 5!Nitro! 6!sulphamoylbenzoðfŁquinoxaline!1\2!dione "NBQX# was

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obtained from Tocris Neuramin "Essex\ U[K[#[ "4R\09S#! "¦#!4!methyl!09\00!dihydro!4H!dibenzoða\dŁcyclohep! ten!4\09!imine hydrogen maleate "MK!790# and 5! chloro!2\3!dihydro!2!"1!norbornen!4!yl#!1H!0\1\3!ben! zothiadiazine!6!sulphonamide 0\0!dioxide "cyclothi! azide# were from RBI "MA\ U[S[A[#[ Other reagents were of the highest purity available from regular commercial sources[ 1[1[ Cell cultures Primary cultures from mouse cerebral cortex were pre! pared as described by Hertz et al[ "0878#[ Brie~y\ cortices were obtained from 04!day old embryos[ The tissue was chopped and dissociated by mild trypsinization "9[919) w:v\ 26>C\ 09 min[# and subsequently triturated in a solu! tion containing DNAse "9[997) w:v# and soybean tryp! sin inhibitor "9[941) w:v#[ The cells were cultured in a modi_ed Dulbecco|s medium "Hertz et al[\ 0871# con! taining 09) "v:v# Fetal calf serum "heat inactivated#\ 13[3 mM KCl\ 9[1 mM L!glutamine\ penicillin "094 units:l#\ insulin "9[0 units:l# and p!aminobenzoic acid "6[2 mM#[ The cells were seeded in NUNC 13!well plates "0[04×095 cells:well# coated with poly!D!lysine "49 mg:l# as described by Sensenbrenner et al[ "0867#[ Cultures were maintained at 26>C in an humidi_ed atmosphere of 4) CO1[ To prevent proliferation of non!neuronal cells in the culture\ 19 mM cytosine arabinofuranoside was added after 37 h in culture[ This treatment reduces the astrocytic contamination to 4Ð09) in the culture "Dichter\ 0867^ Larsson et al[\ 0874#[ Cultures were used for up to 02 days in vitro "DIV#[

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immediately after drug exposure the cells were gently washed with HEPES!bu}ered saline solution and sub! sequently incubated for 04 min with MTT "9[4 mg:ml in HEPES!bu}ered saline solution# at 26>C[ The MTT! solution was removed and the formazan product formed was solubilized in 149 ml of 4) SDS overnight at room temperature[ The absorbance of a 199 ml SDS!aliquot was measured at 449 nm "in an ELISA microplate reader#[ Ten ml of 1!propanol was added to each 199 ml SDS!sample before the A449 was determined in order to remove bubbles[ Viability is expressed as a percentage of control MTT reduction with a baseline correction[ Control values were included in each experiment and represent the quantity of formazan product formed by living cells in the culture in the absence of drug treatment[ 1[3[ Data analysis and statistics The data presented in Fig[ 0 indicate percentage MTT reduction after drug treatment when compared to the control values "099)#[ After exposure of the cultures to 4 mM sodium azide a residual MTT reduction capacity of 08) compared to non!treated controls was observed\ even though when inspected in a phase!contrast micro! scope\ the morphology of the cell cultures revealed that all cells were disintegrated and dead[ Therefore\ results

1[2[ Drug exposure and MTT assay Cultures at various DIV were exposed to drugs for 5 h[ Drug exposure was performed in conditioned medium at 26>C in an humidi_ed atmosphere at 4) CO1[ The _nal concentration of AMPA used was either 14 mM or 499 mM[ Cyclothiazide "49 mM# was added to all incubations with 14 mM AMPA[ Antagonists were added together with AMPA when they were used[ NBQX was dissolved in DMSO such that the _nal concentration in the incu! bation medium was ³9[0) which has no e}ect on the cultures "data not shown#[ Exposure to 4 mM sodium azide was performed in HEPES!bu}ered saline solution "019 mM NaCl\ 4[9 mM KCl\ 9[51 mM MgSO3\ 0[7 mM CaCl1\ 09 mM HEPES\ 5[0 mM glucose^ pH 6[3# for 3 h at 26>C[ Cell viability was measured using the substrate 2!"3\4! dimethylthiazol!1!yl#!1\4!diphenyltetrazolium bromide "MTT# which is reduced by cells with functional mito! chondria to a blue formazan product "Mosmann\ 0872# that can be quanti_ed spectrophotometrically "Slater et al[\ 0852#[ The assay is performed essentially as described by Varming et al[ "0885# with a few modi_cations]

Fig[ 0[ E}ect of cyclothiazide on AMPA!induced neurotoxicity as a function of neuronal development in culture[ Neocortical neurons cul! tured for 1\ 4\ 7 and 01 days in vitro were exposed to 499 mM AMPA "ž# or 14 mM AMPA¦49 mM cyclothiazide "Ž# for 5 h in conditioned medium[ Immediately after exposure\ MTT reduction was determined "Experimental procedures#[ MTT reduction is expressed as a percentage of control in the absence of drug treatment[ The baseline at 08) MTT reduction indicates the point of 099) cell death[ Asterisks indicate statistically signi_cant di}erences "One!way ANOVA]  P  9[991^  P ³ 9[990#[ Each point represents 2Ð6 separate experiments per! formed on di}erent culture preparations and in quadruplicate "n  01Ð 17#[

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expressed as percentage cell death or percentage cell viability "Figs 1Ð4# were corrected for this baseline MTT reduction value[ All data were analysed for statistical signi_cance using the one!way analysis of variance "ANOVA#[ Data were considered signi_cantly di}erent from the control value if P ¾ 9[90[ Values are expressed as means2SEM[

2[ Results 2[0[ Exposure of neocortical neurons to sodium azide The e}ect of sodium azide was examined in cortical neu! rons cultured for 4\ 7 and 09 DIV[ The neurons were exposed to 4 mM sodium azide for 3 h[ As expected\ sodium azide treatment led to a widespread cell death in the cultures but still a residual amount of 06Ð11) MTT was reduced to formazan "4 DIV] 11[220[1)\ 7 DIV]

Fig 1[ Pharmacology of AMPA!induced excitotoxicity in neocortical neurons cultured for 1\ 4\ 7 and 01 DIV[ 499 mM AMPA was exposed to the cultures for 5 h in conditioned medium in the presence of 09 mM MK!790\ 09 mM NBQX or 09 mM NS 283[ The e}ect of the antagonists was determined using the MTT assay "Experimental procedures#[ The results are expressed as percentage cell viability using a baseline cor! rection for the residual 08) MTT reduction in the presence of 4 mM sodium azide[ MK!790 inhibited all AMPA responses seen at 1\ 4 and 7 DIV[ Only at DIV 01 was AMPA¦MK!790 signi_cantly di}erent from control[ AMPA¦NBQX was signi_cantly di}erent compared to control at DIV 7 and 01[ No signi_cant di}erences were observed between AMPA and AMPA¦NBQX at all DIV[ NS 283 did not inhibit the AMPA toxicity produced at DIV 4\ 7 and 01[ NS 283 itself produced toxicity at DIV 1 and DIV 4\ showing signi_cant di}erences from control[ Asterisks indicate statistically signi_cant di}erences from con! trol "One!way ANOVA]  P ³ 9[90^  P ³ 9[990^ P  9[993^ è P  9[991#[ Each point represents 2Ð6 separate experiments per! formed on di}erent culture preparations and in quadruplicate "n  01Ð 17#[

Fig[ 2[ E}ect of cyclothiazide on AMPA!induced excitotoxicity in neo! cortical neurons cultured for 1\ 4\ 7 and 01 DIV[ The pharmacology of the induced excitotoxicity was examined using di}erent antagonists] 09 mM MK!790\ 09 mM NBQX or 09 mM NS 283[ 14 mM AMPA and 49 mM cyclothiazide were incubated in conditioned medium for 5 h in the presence of an antagonist[ MK!790 and NS 283 block part of the AMPA induced cell death at DIV 4\ 7 and 01\ while NBQX prevented toxicity at all DIV "no signi_cant di}erences from control#[ Toxicity was assessed using the MTT assay "Experimental procedures#[ Cell viability is expressed as a percentage of control in absence of drug treatment and corrected for the residual 08) MTT reduction seen upon sodium azide exposure "Experimental procedures and Results#[ Asterisks indicate statistically signi_cant di}erences "One!way ANOVA]  P ³ 9[990#[ Each point represents 2Ð5 separate experi! ments performed on di}erent culture preparations and in quadruplicate "n  01Ð17#[

06[629[3) and 09 DIV] 06[920[6)#[ Morphological examination of the neurons\ however\ showed extensive cell death with no intact neurons left in the culture[ Exposure of DIV 4 and 02 neurons to 4 mM sodium azide in conditioned media for 5 h produced similar MTT reduction as a 3 h incubation "data not shown#[ For this reason\ results expressed as cell viability or cell death were calculated using 08) MTT reduction as the baseline corresponding to 099) cell death[ The 08) MTT reduction is the mean value of results from all DIV\ since no signi_cant di}erence appears between the di}erent DIV upon exposure to sodium azide "one!way ANOVA\ P − 9[933#[ 2[1[ Effect of cyclothiazide on AMPA mediated toxicity The developmental pro_le of AMPA receptor mediated toxicity was examined in cerebral cortical neurons using the MTT assay for determination of cell viability[ Exposure of cortical neurons to 499 mM AMPA for 5 h produced increasing toxicity after DIV 4 as shown in Fig[ 0[ No toxicity was seen at DIV 1[ However\ only a

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Fig[ 3[ E}ect of 1\2!benzodiazepines on the excitotoxicity induced by 499 mM AMPA in neocortical neurons cultured for 1\ 4\ 7 and 01 DIV[ The pharmacology of the induced excitotoxicity was examined using three di}erent antagonists] 49 mM GYKI 41355\ 49 mM GYKI 42394 or 49 mM GYKI 42544[ 499 mM AMPA was incubated in conditioned medium for 5 h in the presence of an antagonist[ Toxicity was assessed using the MTT assay[ Cell viability is expressed as a percentage of control in absence of drug treatment and corrected for the residual 08) MTT reduction seen upon sodium azide exposure "Experimental procedures and Results#[ All GYKI compounds antagonized AMPA! induced cell death at all DIV except for neurons cultured for 01 DIV[ Asterisks indicate statistically signi_cant di}erences "One!way ANOVA]  P ³ 9[90^  P ³ 9[990#[ Each point represents 2Ð6 separate experiments performed on di}erent culture preparations and in quad! ruplicate "n  01Ð17#[

maximum of 30) cell death was observed with 499 mM AMPA when neurons were cultured for 01 DIV[ To test if this rather modest e}ect of 499 mM AMPA was due to desensitization of the AMPA receptors\ cultures were incubated with 14 mM AMPA for 5 h in the presence of 49 mM cyclothiazide[ As can be seen in Fig[ 0\ cyclothiazide strongly potentiated the neurotoxic response at a lower concentration of AMPA[ AMPA¦cyclothiazide resulted in a maximal toxicity "66)# found at DIV 01 which was almost twice as large as that found in the absence of cyclothiazide[ Cyclothiazide itself did not induce any sig! ni_cant di}erences from control values when applied to neurons for 5 h at all DIV|s[ It was observed that 499 mM AMPA¦49 mM cyclothiazide produced the same developmental pro_le of neurotoxicity as 14 mM AMPA¦49 mM cyclothiazide "data not shown# but that the toxic action was more pronounced at DIV 7 and 01[ 2[2[ AMPA receptor mediated toxicity*EAA antagonist pharmacology In order to be able to di}erentiate between NMDA and AMPA receptor mediated toxicity\ the EAA antagonists]

498

Fig[ 4[ E}ect of 1\2!benzodiazepines on the excitotoxicity induced by 14 mM AMPA in neocortical neurons cultured for 1\ 4\ 7 and 01 DIV when desensitization is blocked by cyclothiazide[ The pharmacology of the induced excitotoxicity was examined using three di}erent antag! onists] 49 mM GYKI 41355\ 49 mM GYKI 42394 or 49 mM GYKI 42544[ 14 mM AMPA and 49 mM cyclothiazide were incubated in conditioned medium for 5 h in the presence of an antagonist[ Toxicity was assessed using the MTT assay "Experimental procedures#[ Cell viability is expressed as a percentage of control in absence of drug treatment and corrected for the residual 08) MTT reduction seen upon sodium azide exposure "Experimental procedures and Results#[ 49 mM GYKI 41355 was not able to antagonize AMPA!induced toxicity at DIV 4\ 7 and 01\ whereas GYKI 42394 blocked toxicity at DIV 4\ but not at DIV 7 and 01[ GYKI 42544 was the most potent antagonist\ inhibiting all induced toxicity at all DIV[ Asterisks indicate statistically signi_cant di}erences "One!way ANOVA]  P ³ 9[90^  P ³ 9[990#[ Each point represents 2Ð4 separate experiments performed on di}erent culture preparations and in quadruplicate "n  01Ð19#[

MK!790\ NBQX and NS 283 were included in the incu! bation media together with 499 mM AMPA "Fig[ 1# or 14 mM AMPA¦49 mM cyclothiazide "Fig[ 2#[ MK!790 and NBQX were non!toxic to the neurons since no di}erence from control was observed when the antag! onists were applied alone in the incubation media "data not shown#[ However\ NS 283 was found to be toxic to the neurons when cultured for 1 and 4 DIV "P ³ 9[990\ n  12Ð13# but was non!toxic at DIV 7 and 01[ The selective NMDA receptor antagonist MK!790 "09 mM# prevented 499 mM AMPA toxicity at all DIV except for DIV 01 "Fig[ 1#[ However\ when 49 mM cyclothiazide was added to prevent desensitization of the AMPA recep! tor channels\ MK!790 only partially blocked the AMPA response seen in neurons cultured at all DIV except DIV 1 "Fig[ 2#[ NBQX "09 mM#\ a competitive non!NMDA receptor antagonist\ completely antagonized the response observed with 14 mM AMPA¦49 mM cyclothiazide at all DIV "Fig[ 2# but did not block the responses seen with 499 mM AMPA at DIV 4\ 7 and 01 "Fig[ 1#[ Here it is seen that 09 mM NS 283 did not block 499 mM AMPA

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"Fig[ 1# but antagonized the AMPA!mediated toxicity when desensitization was blocked "Fig[ 2#\ although less e.ciently than 09 mM NBQX[ 2[3[ Antagonism by GYKI compounds Here it is observed that the GYKI compounds prevented most AMPA!mediated toxicity in cortical neurons[ When 499 mM AMPA became toxic to neurons cultured for 7 DIV\ 49 mM GYKI 41355 and 49 mM GYKI 42544 totally prevented neurotoxicity\ whereas 49 mM GYKI 42394 was insu.cient to block all toxicity "Fig[ 3#[ Cell death of DIV 01 cultures exposed to 499 mM AMPA could not be completely blocked by the GYKI compounds[ Of these compounds\ GYKI 42544 was the most potent antagonist when cells were exposed to 14 mM AMPA¦49 mM cyclo! thiazide "Fig[ 4#[ Fifty mM GYKI 42544 prevented tox! icity at all DIV\ whereas 49 mM GYKI 41355 and 49 mM GYKI 42394 could only sub!maximally protect "Fig[ 4#[ GYKI 41355 and GYKI 42544 were non!toxic to the neurons when applied alone to the cultures\ but GYKI 42394 showed signi_cant di}erences from the control value when applied to the cultures at DIV 1 "P ¾ 9[995\ n  13# and 4 "P ¾ 9[992\ n  19#[

3[ Discussion 3[0[ AMPA toxicity in neocortical neurons Exposing neocortical neurons at di}erent DIV to 499 mM AMPA for 5 h showed that AMPA toxicity develops late in culture and that it was only possible to produce 30) cell death\ as measured by the ability of functional cells to reduce MTT[ This is in agreement with Larm et al[ "0885# who have shown that 13 h exposure to 099 mM AMPA produced 49) cell death in neocortical neurons cultured for 5 days[ Also\ the observed developmental pro_le of AMPA excitotoxicity relates well to previous _ndings "Frandsen and Schousboe\ 0889#[ However\ a high concentration of AMPA "499 mM# produced rela! tively little cell death which does not correspond with other reports using lactate dehydrogenase "LDH# leakage as the viability test "Frandsen and Schousboe\ 0876\ 0889^ Koh et al[\ 0889#[ These studies have shown that pro! longed exposure to a low concentration of AMPA pro! duced widespread cell death in cultured cerebral cortical neurons[ This disagreement with reported results could re~ect the di}erence in the assay used to assess cell viability[ A measurement of LDH released from neurons will also take into account living cells with a compromised cell membrane[ In this context\ LDH measurement may over!estimate the neurotoxicity produced[ When using the MTT assay\ however\ we measure neurons that are able to reduce MTT in the mitochondria "Slater et al[\ 0852# and in the cytosol "Berridge and Tan\ 0882# and

presumably only neurons that are able to take up MTT by endocytosis "Liu et al[\ 0886#[ This should re~ect a better de_nition of cell death since lack of endocytosis and reduction of MTT will indicate neurons without the capability to generate ATP and maintain metabolism[ Experiments with 4 mM sodium azide were performed to validate the MTT assay[ Sodium azide inhibits the respiratory chain at complex IV\ giving rise to a lack of neuronal mitochondrial function and subsequent cell death[ Since the reduction of MTT occurs at an earlier point in the electron transport chain "Berridge and Tan\ 0882#\ azide does not directly inhibit this reaction[ When exposing neurons cultured for 4\ 7 and 09 DIV to sodium azide\ a residual capacity to reduce MTT of 08) was found[ However\ the morphology of the culture clearly showed disintegrated and dead neurons[ The 08) residual MTT reduced may be due to an enzymatic reduction of MTT by non!mitochondrial dehydrogenases "Berridge and Tan\ 0882#[ 3[1[ Cyclothiazide enhances AMPA toxicity in neocortical neurons It is shown here that AMPA receptor mediated exci! totoxicity is highly dependent upon receptor desen! sitization[ By blocking receptor desensitization with cyclothiazide\ the toxicity produced by AMPA in the neocortical neurons is increased and also a clear devel! opmental increase in AMPA mediated neurotoxicity is observed[ The potentiation of AMPA excitotoxicity when receptor desensitization is blocked is in agreement with other reports where it was found that prolonged exposure of rat hippocampal neurons "May and Robison\ 0882# or cerebellar granule cells "Hack et al[\ 0884^ Cebers et al[\ 0886^ Impagnatiello et al[\ 0886# to a high concentration of AMPA produced little or no neurotoxicity but that co!incubation with cyclothiazide highly enhanced the toxicity produced by AMPA[ Reports showing that the intracellular free calcium concentration and 34Ca1¦ uptake into cultured cortical neurons and cerebellar gran! ule cells were increased when AMPA receptor desen! sitization was blocked "Cebers and Liljequist\ 0884^ Hoyt et al[\ 0884^ Savidge and Bristow\ 0886# support our _n! dings since the increased intracellular calcium con! centration stimulated by excitatory amino acids has been shown to be associated with neurotoxicity "Choi\ 0874^ Schousboe et al[\ 0880#[ Calcium in~ux through AMPA receptor channels is thought to be highly dependent on the presence of the GluR1 subunit in the receptor com! plex "Burnashev et al[\ 0881#[ Since the amount of GluR1"R# subunit relative to the other GluR subunits will determine the calcium permeability\ it can be pre! dicted that neurons having little or no expression of GluR1"R# will possess high calcium permeability[ It would therefore be interesting to examine the expression of the GluR1 subunit relative to GluR0\ GluR2 and

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GluR3 during culture development\ considering that AMPA mediated toxicity is increasing during devel! opment[ However\ since desensitization plays a con! siderable role for AMPA mediated toxicity\ the kinetics of the receptor complex may be even more important for the functional pro_le of the AMPA receptor[ 3[2[ Pharmacology of AMPA mediated neurotoxicity AMPA toxicity produced in the cortical neurons is mediated partly by the NMDA receptors since the NMDA antagonist MK!790 is able to prevent all or some toxicity when applied together with AMPA or AMPA ¦cyclothiazide[ That NMDA receptor antagonists are able to partially inhibit the toxicity produced by AMPA in the presence of cyclothiazide is in agreement with Impagnatiello et al[ "0886# who showed that dizocilpine was able to prevent some AMPA induced neurotoxicity in the presence of cyclothiazide[ However\ other reports have shown no e}ect of NMDA receptor antagonists on AMPA mediated toxicity under non!desensitizing con! ditions "Hack et al[\ 0884^ Cebers et al[\ 0886#[ The {indirect| AMPA toxicity could be due to release of glu! tamate or aspartate either when the cell membrane is depolarized as monovalent ions ~ow through AMPA receptor channels upon receptor stimulation\ or release of glutamate from dead and dying cells[ Recently it has been shown that stimulation of AMPA:kainate receptors by domoic acid can cause non!vesicular EAA release in cerebellar granule neurons by reversal of the high!a.nity glutamate transporter "Berman and Murray\ 0886#[ Such mechanisms may also operate in cortical neuronal cul! tures "Dugan et al[\ 0884#[ As the cell membrane is depo! larized the magnesium block of the NMDA receptor channels is released and then subsequent activation of NMDA channels by released glutamate or aspartate can occur[ When cultures are exposed to 499 mM AMPA\ MK!790 was able to prevent cell death except from neu! rons cultured for 01 DIV\ where some toxicity was still observed "01)#[ This suggests that most neurotoxicity produced by 499 mM AMPA is due to an indirect acti! vation of the NMDA receptors but that in mature cul! tures "DIV 01# 01) cell death is mediated directly through AMPA receptor channels "Fig[ 1#[ However\ when receptor desensitization is blocked the direct AMPA mediated toxicity is high "½59) at DIV 01# since MK!790 only blocked 09Ð19) of the AMPA response "Fig[ 2# but 09 mM NBQX\ a non!NMDA receptor antag! onist\ completely blocked toxicity[ Yet\ 09 mM NBQX was insu.cient to inhibit the response to 499 mM AMPA "Fig[ 1#\ probably re~ecting that a higher concentration of the competitive antagonist is necessary to completely antagonize the e}ect of 499 mM AMPA[ Partial inhibition by 09 mM NS 283 was seen of the toxicity of AMPA under non!desensitizing conditions at all DIV "Fig[ 2#[ Since NS 283 has been reported to have

400

a slight selectivity for kainate receptors "Paternain et al[\ 0885# there was a possibility that this antagonist could be used as a kainate receptor antagonist[ Unfortunately\ since NS 283 was observed here to block part of the non! desensitizing AMPA response\ this antagonist could not be used to di}erentiate between AMPA and kainate receptors[ In addition\ NS 283 exhibited some neuro! toxicity\ complicating its use as an antagonist[ The 1\2!benzodiazepines are reported to be fairly selec! tive non!competitive antagonists for the AMPA recep! tors\ being active at the kainate receptor subtypes only at higher concentrations "Donevan and Rogawski\ 0882^ Paternain et al[\ 0884^ Vizi et al[\ 0885#[ Here we have shown that the 1\2!benzodiazepines are potent antag! onists of the AMPA response "Figs 3 and 4#[ The most selective and potent 1\2!benzodiazepine antagonist for the AMPA receptors\ GYKI 42544 "Bleakman et al[\ 0885^ Kovacs and Szabo\ 0886#\ blocked all observed AMPA responses except for 499 mM AMPA at DIV 01 "½09) cell death was seen#[ As with NBQX\ this may re~ect an insu.ciently high concentration of the antag! onist for complete receptor blockade[ The potency rank order of the GYKI compounds was found to be] GYKI 42544 × GYKI 41355 − GYKI 42394 for activity under both desensitizing and non!desensitizing conditions[ This potency order at AMPA receptors is in agreement with what has been seen both in vitro "Bleakman et al[\ 0885# and in vivo "Donevan et al[\ 0883#[ We have shown that prolonged exposure time with AMPA mediates increasing neurotoxicity when receptor desensitization is blocked by cyclothiazide[ The direct component of AMPA receptor mediated toxicity could be due to cell membrane depolarization and subsequent calcium in~ux through voltage!gated calcium channels\ reversal of the Na¦!Ca1¦ exchanger and:or increased calcium in~ux through calcium permeable AMPA recep! tor channels when receptor desensitization is blocked[ The indirect component of AMPA receptor mediated toxicity involves activation of the NMDA receptor chan! nels\ likely as a consequence of glutamate or aspartate being released from neurons by AMPA receptor mediated depolarization[ The exact mechanism involved in direct AMPA receptor mediated toxicity is still unknown and is under further investigation[ The early excitotoxicity produced by excitatory amino acid agonists has been believed to be mediated via a necrotic cell death\ inducing acute organelle swelling and a delayed neuronal disintegration[ AMPA was reported not to mediate acute swelling but\ upon prolonged exposure\ to mediate a delayed neuronal disintegration which showed a dependency on extracellular calcium "Koh et al[\ 0889^ Choi\ 0881#[ Other mechanisms may also be involved in AMPA mediated cell death since it has been reported that prolonged "13 h# exposure to high concentrations of AMPA induces neuronal death in cultured murine cortical neurons via apoptosis "Larm et

401

J[B[ Jensen et al[:Neurochem[ Int[ 21 "0887# 494Ð402

al[\ 0886#[ Interestingly\ this e}ect was blocked by non! NMDA receptor antagonists but not by the NMDA receptor antagonist MK!790\ implying a neurotoxicity that is mediated directly via AMPA receptors[ The direct AMPA mediated toxicity could be a consequence of cell membrane depolarization and secondary calcium in~ux through voltage!gated calcium channels\ reversal of the Na¦!Ca1¦ exchanger or direct calcium in~ux through AMPA receptor channels[ Yet it is not simply the calcium in~ux per se that induces toxicity since it has been shown that high potassium concentration "49 mM# does not induce cell death in neocortical neurons "Frandsen and Schousboe\ 0880#[ Nevertheless\ the in~uence of voltage! gated calcium channels in AMPA mediated toxicity can! not be totally excluded since it has been reported that voltage!gated calcium channel blockers attenuate the AMPA induced rise in intracellular calcium con! centration and cell death "Weiss et al[\ 0889^ Frandsen and Schousboe\ 0881#[ A prolonged activation of AMPA receptors and persistent calcium in~ux when desen! sitization is blocked could alter the calcium homeostasis of the neuron and subsequently lead to cell death[ Both necrosis and apoptosis can occur after loss of intracellular calcium homeostasis "Choi\ 0881# implying that AMPA receptors may be important in glutamate receptor mediated cell death[ The physiological implications of the present _ndings are that a rapid desensitization of AMPA receptors under normal conditions probably pro! tects neurons against excessive glutamate stimulation and thereby against cell death[ Under pathological conditions however\ the possibility exists that toxicity is mediated directly through the AMPA receptor channels[

Acknowledgements We are grateful to Dr P[ Krogsgaard!Larsen and Dr U[ Madsen\ The Royal Danish School of Pharmacy\ for providing us with AMPA^ to J[ Drejer\ NeuroSearch A:S\ for providing us with NS 283 and to Dr P[ Somogyi\ Institute for Drug Research\ Hungary\ for providing the GYKI compounds[ The study was supported by grants from the Danish State Biotechnology Programs\ the Danish Medical Research Council "8699650# and the Lundbeck Foundation[ D[S[P[ is supported by an Alfred Benzon Foundation fellowship[

References Angulo\ M[C[\ Lambolez\ B[\ Audinat\ E[\ Hestrin\ S[\ Rossier\ J[\ 0886[ Subunit composition\ kinetic\ and permeation properties of AMPA receptors in single neocortical nonpyramidal cells[ J[ Neurosci[ 06\ 5574Ð5585[ Berman\ F[W[\ Murray\ T[F[\ 0886[ Domoic acid neurotoxicity in cul! tured cerebellar granule neurons is mediated predominantly by

NMDA receptors that are activated as a consequence of excitatory amino acid release[ J[ Neurochem[ 58\ 582Ð692[ Berridge\ M[V[\ Tan\ A[S[\ 0882[ Characterization of the cellular reduction of 2!"3\4!dimethylthiazol!1!yl#!1\4!diphenyltetrazolium bromide "MTT#] subcellular localization\ substrate dependence\ and involvement of mitochondrial electron transport in MTT reduction[ Arch[ Biochem[ Biophys[ 292\ 363Ð371[ Bleakman\ D[\ Ballyk\ B[A[\ Schoepp\ D[D[\ Palmer\ A[J[\ Bath\ C[P[\ Sharpe\ E[F[\ Woolley\ M[L[\ Bufton\ H[R[\ Kamboj\ R[K[\ Tarnawa\ I[\ Lodge\ D[\ 0885[ Activity of 1\2!benzodiazepines at native rat and recombinant human glutamate receptors in vitro] stereospeci_city and selectivity pro_les[ Neuropharmacol[ 24\ 0578Ð 0691[ Burnashev\ N[\ 0882[ Recombinant ionotropic glutamate receptors] functional distinctions imparted by di}erent subunits[ Cell Physiol[ Biochem[ 2\ 207Ð220[ Burnashev\ N[\ Monyer\ H[\ Seeburg\ P[H[\ Sakmann\ B[\ 0881[ Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit[ Neuron 7\ 078Ð087[ Cebers\ G[\ Liljequist\ S[\ 0884[ Modulation of AMPA:kainate recep! tors by cyclothiazide increases cytoplasmic free Ca1¦ and 34Ca1¦ uptake in brain neurons[ Eur[ J[ Pharmacol[ 189\ 094Ð004[ Cebers\ G[\ Zhivotovsky\ B[\ Ankarcrona\ M[\ Liljequist\ S[\ 0886[ AMPA neurotoxicity in cultured cerebellar granule neurons] mode of cell death[ Brain Res[ Bull[ 32\ 282Ð392[ Choi\ D[W[\ 0874[ Glutamate neurotoxicity in cortical cell culture is calcium dependent[ Neurosci[ Lett[ 47\ 182Ð186[ Choi\ D[W[\ 0881[ Excitotoxic cell death[ J[ Neurobiol[ 12\ 0150Ð0165[ Choi\ D[W[\ Koh\ J[!Y[\ Peters\ S[\ 0877[ Pharmacology of glutamate neurotoxicity in cortical cell culture] attenuation by NMDA antag! onists[ J[ Neurosci[ 7\ 074Ð085[ Dichter\ M[A[\ 0867[ Rat cortical neurons in culture] methods\ cell morphology\ electrophysiology and synapse formation[ Brain Res[ 038\ 168Ð182[ Donevan\ S[D[\ Rogawski\ M[A[\ 0882[ GYKI 41355\ a 1\2!benzo! diazepine\ is a highly selective\ noncompetitive antagonist of AMPA:kainate receptor responses[ Neuron 09\ 40Ð48[ Donevan\ S[D[\ Yamaguchi\ S!I[\ Rogawski\ M[A[\ 0883[ Non!N! methyl!D!aspartate receptor antagonism by 2!N!substituted 1\2! benzodiazepines] relationship to anticonvulsant activity[ J[ Phar! macol[ Exp[ Therap[ 160\ 14Ð18[ Dugan\ L[L[\ Bruno\ V[M[G[\ Amagasu\ S[M[\ Gi}ard\ R[G[\ 0884[ Glia modulate the response of murine cortical neurons to exci! totoxicity] Glia exacerbate AMPA neurotoxicity[ J[ Neurosci[ 04\ 3434Ð3444[ Frandsen\ A[\ Schousboe\ A[\ 0876[ Time and concentration depen! dency of the toxicity of excitatory amino acids on cerebral neurons in primary culture[ Neurochem[ Int[ 09\ 472Ð480[ Frandsen\ A[\ Schousboe\ A[\ 0889[ Development of excitatory amino acid induced cytotoxicity in cultured neurons[ Int[ J[ Devl[ Neurosci[ 7\ 198Ð105[ Frandsen\ A[\ Schousboe\ A[\ 0880[ Dantrolene prevents glutamate cytotoxicity and Ca1¦ release from intracellular stores in cultured cerebral cortical neurons[ J[ Neurochem[ 45\ 0964Ð0967[ Frandsen\ A[\ Schousboe\ A[\ 0881[ Mobilization of dantrolene!sen! sitive intracellular calcium pools is involved in the cytotoxicity induced by quisqualate and N!methyl!D!aspartate but not by 1! amino!2!"2!hydroxy!4!methylisoxazol!3!yl#propionate and kainate in cultured cerebral cortical neurons[ Proc[ Natl[ Acad[ Sci[ U[S[A[ 78\ 1489Ð1483[ Geiger\ J[R[P[\ Melcher\ T[\ Koh\ D[!S[\ Sakman\ B[\ Seeburg\ P[H[\ Jonas\ P[\ Monyer\ H[\ 0884[ Relative abundance of subunit mRNAs determines gating and Ca1¦ permeability of AMPA receptors in principal neurons and interneurons in rat CNS[ Neuron 04\ 082Ð 193[ Hack\ N[J[\ Sluiter\ A[A[\ Balazs\ R[\ 0884[ AMPA receptors in cer!

J[B[ Jensen et al[:Neurochem[ Int[ 21 "0887# 494Ð402 ebellar granule cells during development in culture[ Dev[ Brain Res[ 76\ 44Ð50[ Hertz\ E[\ Yu\ A[C[H[\ Hertz\ L[\ Juurlink\ B[H[J[\ Schousboe\ A[\ 0878[ Preparation of primary cultures of mouse cortical neurons[ In Shahar\ A[\ de Vellis\ J[\ Vernadakis\ A[\ Haber\ B[ "Eds#\ A dis! section and tissue culture manual of the nervous system\ chapter 39[ Alan R[ Liss[ Inc\ New York\ pp[ 072Ð075[ Hertz\ L[\ Juurlink\ B[H[J[\ Fosmark\ H[\ Schousboe\ A[\ 0871[ Astro! cytes in primary cultures[ In Pfei}er\ K[S[E[ "Ed[#\ Neuroscience approached through cell culture\ Vol[ 0\ CRC Press\ Boca Raton\ Fl\ U[S[A[ pp[ 064Ð075[ Hollmann\ M[\ Hartley\ M[\ Heinemann\ S[\ 0880[ Ca1¦ permeability of KA!AMPA!gated glutamate receptor channels depends on subunit composition[ Science 141\ 740Ð742[ Hoyt\ K[R[\ Rajdev\ S[\ Fattman\ C[L[\ Reynolds\ I[J[\ 0884[ Cyclo! thiazide modulates AMPA receptor!mediated increases in intra! cellular free Ca1¦ and Mg1¦ in cultured neurons from rat brain[ J[ Neurochem[ 53\ 1938Ð1945[ Impagnatiello\ F[\ Oberto\ A[\ Longone\ P[\ Costa\ E[\ Guidotti\ A[\ 0886[ 6!cloro!2!methyl!2\3!dihydro!1H!0\1\3!benzothiadiazine S\S! dioxide] a partial modulator of AMPA receptor desensitization devoid of neurotoxicity[ Proc[ Natl[ Acad[ Sci[ U[S[A[ 83\ 6942Ð 6947[ Kim\ J[P[\ Koh\ J[\ Choi\ D[W[\ 0876[ L!Homocysteate is a potent neurotoxin on cultured cortical neurons[ Brain Res[ 326\ 092Ð009[ Kiskin\ N[I[\ Krishtal\ O[A[\ Tsyndrenko\ A[Y[\ 0875[ Excitatory amino acid receptors in hippocampal neurons] kainate fails to desensitize them[ Neurosci[ Lett[ 52\ 114Ð129[ Koh\ J[!Y[\ Goldberg\ M[P[\ Hartley\ D[M[\ Choi\ D[W[\ 0889[ Non! NMDA receptor!mediated neurotoxicity in cortical culture[ J[ Neurosci[ 09\ 582Ð694[ Kovacs\ A[D[\ Szabo\ G[\ 0886[ GYKI 42544\ a 1\2!benzodiazepine\ non!competitively protects cultured neurons against AMPA toxicity[ Eur[ J[ Pharmacol[ 220\ 82Ð85[ Lambolez\ B[\ Ropert\ N[\ Perrais\ D[\ Rossier\ J[\ Hestrin\ S[\ 0885[ Correlation between kinetics and RNA splicing of a!amino!2!hy! droxy!4!methylisoxazole!3!propionic acid receptors in neocortical neurons[ Proc[ Natl[ Acad[ Sci[ U[S[A[ 82\ 0686Ð0791[ Larm\ J[A[\ Cheung\ N[S[\ Beart\ P[M[\ 0885[ "S#!4!~uorowillardiine! mediated neurotoxicity in cultured murine cortical neurons occurs via AMPA and kainate receptors[ Eur[ J[ Pharmacol[ 203\ 138!143[ Larm\ J[A[\ Cheung\ N[S[\ Beart\ P[M[\ 0886[ Apoptosis induced via AMPA!selective glutamate receptors in cultured murine cortical neurons[ J[ Neurochem[ 58\ 506Ð511[ Larsson\ O[M[\ Drejer\ J[\ Kvamme\ E[\ Svenneby\ G[\ Hertz\ L[\ Sch! ousboe\ A[\ 0874[ Ontogenetic development of glutamate and GABA metabolizing enzymes in cultured cerebral cortex inter! neurons and in cerebral cortex in vivo[ Int[ J[ Devl[ Neurosci[ 2\ 066Ð074[ Liu\ Y[\ Peterson\ D[A[\ Kimura\ H[\ Schubert\ D[\ 0886[ Mechanism of cellular 2!"3\4!dimethylthiazol!1!yl#!1\4!diphenyltetrazolium bromide "MTT# reduction[ J[ Neurochem[ 58\ 4700Ð4482[ Lomeli\ H[\ Mosbacher\ J[\ Melcher\ T[\ Hoger\ T[\ Geiger\ J[R[P[\ Kuner\ T[\ Monyer\ H[\ Higuchi\ M[\ Bach\ A[\ Seeburg\ P[\ 0883[ Control of kinetic properties of AMPA receptor channels by nuclear RNA editing[ Science 155\ 0698Ð0602[ May\ P[C[\ Robison\ P[M[\ 0882[ Cyclothiazide treatment unmasks AMPA excitotoxicity in rat primary hippocampal cultures[ J[ Neu! rochem[ 59\ 0060Ð0063[ Mosbacher\ J[\ Schoepfer\ R[\ Monyer\ H[\ Burnashev\ N[\ Seeburg\ P[H[\ Ruppersberg\ J[P[\ 0883[ A molecular determinant for sub! millisecond desensitization in glutamate receptors[ Science 155\ 0948Ð0951[ Mosman\ T[\ 0872[ Rapid colorimetric assay for cellular growth and

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survival] application to proliferation and cytotoxicity assays[ J[ Immun[ Meth[ 54\ 44Ð52[ Moudy\ A[M[\ Yamada\ K[A[\ Rothman\ S[M[\ 0883[ Rapid desen! sitization determines the pharmacology of glutamate neurotoxicity[ Neuropharmacol[ 22\ 842Ð851[ Olney\ J[W[\ 0877[ Endogenous excitotoxins and neuropathological disorders[ In Lodge\ D[ "Ed[#\ Excitatory Amino Acids in Health and Disease\ Wiley\ London\ pp[ 186Ð219[ Partin\ K[M[\ Fleck\ M[W[\ Mayer\ M[L[\ 0885[ AMPA receptor ~ip: ~op mutants a}ecting deactivation\ desensitization\ and modulation by cyclothiazide\ aniracetam\ and thiocyanate[ J[ Neurosci[ 05\ 5523Ð5536[ Paternain\ A[V[\ Morales\ M[\ Lerma\ J[\ 0884[ Selective antagonism of AMPA receptor unmasks kainate receptor!mediated responses in hippocampal neurons[ Neuron 03\ 074Ð078[ Paternain\ A[V[\ Vicente\ A[\ Nielsen\ E[O š[\ Lerma J[\ 0885[ Com! parative antagonism of kainate!activated kainate and AMPA recep! tors in hippocampal neurons[ Eur[ J[ Neurosci[ 7\ 1018Ð1025[ Patneau\ D[K[\ Mayer\ M[L[\ 0880[ Kinetic analysis of interactions between kainate and AMPA] evidence for activation of a single receptor in mouse hippocampal neurons[ Neuron 5\ 674Ð687[ Savidge\ J[R[\ Bristow\ D[R[\ 0886[ Distribution of Ca1¦!permeable AMPA receptors among cultured rat cerebellar granule cells[ Neuro Report 7\ 0766Ð0771[ Schousboe\ A[\ Frandsen\ A[\ Wahl\ P[\ Krogsgaard!Larsen\ P[\ 0880[ Excitatory amino acid induced cytotoxicity in cultured neurons] role of intracellular Ca1¦ homeostasis[ In Ascher\ P[\ Choi\ D[W[\ Christen\ Y[ "Eds#\ Glutamate\ Cell Death and Memory\ Springer! Verlag\ Berlin\ Heidelberg\ pp[ 026Ð041[ Sensenbrenner\ M[\ Maderspach\ K[\ Latzkovits\ L[\ Jaros\ G[\ 0867[ Neuronal cells from chick embryo cerebral hemispheres cultivated on polylysine!coated surfaces[ Devl[ Neurosci[ 0\ 80Ð090[ Sheardown\ M[J[\ Nielsen\ E[O š[\ Hansen\ A[J[\ Jacobsen\ P[\ Honore\ T[\ 0889[ 1\2!dihydroxy!5!nitro!6!sulfamoyl!benzo"F#quinoxaline] a neuroprotectant for cerebral ischemia[ Science 136\ 460Ð463[ Slater\ T[F[\ Sawyer\ B[\ Strauli U[\ 0852[ Studies on succinate!tetra! zolium reductase systems[ III[ Points of coupling of four di}erent tetrazolium salts[ Biochim Biophys[ Acta 66\ 272Ð282[ Swanson\ G[T[\ Kamboj\ S[K[\ Cull!Candy\ S[G[\ 0886[ Single!channel properties of recombinant AMPA receptors depend on RNA edit! ing\ splice variation\ and subunit composition[ J[ Neurosci[ 06\ 47Ð 58[ Tang\ C[!M[\ Shi\ Q[!Y[\ Katchman\ A[\ Lynch\ G[\ 0880[ Modulation of the time course of fast EPSCs and glutamate channel kinetics by aniracetam[ Science 143\ 177Ð189[ Thio\ L[L[\ Cli}ord\ D[B[\ Zorumski\ C[F[\ 0881[ Blockade of ion! otropic quisqualate receptor desensitization by wheat germ agglu! tinin in cultured postnatal rat hippocampal neurons[ J[ Neurophysiol[ 57\ 0806Ð0818[ Varming\ T[\ Drejer\ J[\ Frandsen\ A[\ Schousboe\ A[\ 0885[ Charac! terization of a chemical anoxia model in cerebellar granule neurons using sodium azide] protection by nifedipine and MK!790[ J[ Neuro! sci[ Res[ 33\ 39Ð35[ Vizi\ E[S[\ Mike\ A[\ Tarnawa\ I[\ 0885[ 1\2!Benzodiazepines "GYKI 41355 and analogs#] negative allosteric modulators of AMPA recep! tors[ CNS Drug Reviews 1\ 80Ð015[ Weiss\ J[H[\ Hartley\ D[M[\ Koh\ J[\ Choi\ D[W[\ 0889[ The calcium channel blocker nifedipine attenuates slow excitatory amino acid neurotoxicity[ Science 136\ 0363Ð0366[ Wong\ E[H[\ Kemp\ J[A[\ Priestley\ T[\ Knight\ A[R[\ Woodru}\ G[N[\ Iversen L[L[\ 0875[ The anticonvulsant MK!790 is a potent N! methyl!D!aspartate antagonist[ Proc[ Natl[ Acad[ Sci[ U[S[A[ 72\ 6093Ð6097[ Yamada\ K[A[\ Tang\ C[!M[\ 0882[ Benzothiadiazides inhibit rapid glutamate receptor desensitization and enhance glutamatergic syn! aptic currents[ J[ Neurosci[ 02\ 2893Ð2804[