Proc. Natl. Acad. Sci. USAVol. 93, pp. 2312-2316, March 1996Neurobiology

,3-Amyloid toxicity in organotypic hippocampal cultures:Protection by EUK-8, a synthetic catalytic free radical scavengerANNADORA J. BRUCE*t, BERNARD MALFROYt, AND MICHEL BAUDRY*

*Neuroscience Program, University of Southern California, Los Angeles, CA 90089-2520; and *Eukarion, Inc., Bedford, MA 01730

Communicated by Richard F. Thompson, University of Southern California, Los Angeles, CA, November 14, 1995 (received for reviewAugust 7, 1995)

ABSTRACT Oxygen free radicals have been proposed tomediate amyloid peptide (I3AP)-induced neurotoxicity. To testthis hypothesis, we evaluated the effects of EUK-8, a syntheticcatalytic superoxide and hydrogen peroxide scavenger, onneuronal injury produced by 8AP in organotypic hippocam-pal slice cultures. Cultures of equivalent postnatal day 35(defined as mature) and 14 (defined as immature) wereexposed to various concentrations of ,IAP (1-42 or 1-40) inthe absence or presence of 25 ,uM EUK-8 for up to 72 hours.Neuronal injury was assessed by lactate dehydrogenase re-lease and semiquantitative analysis of propidium iodide up-take at various times after the initiation of.8AP exposure. Freeradical production was inferred from the relative increase indichlorofluorescein fluorescence, and the degree of lipidperoxidation was determined by assaying thiobarbituric acid-reactive substances. Treatment of mature cultures with .AP(50-250 jig/ml) in serum-free conditions resulted in a re-producible pattern of damage, causing a time-dependentincrease in neuronal injury accompanied with formation ofreactive oxygen species. However, immature cultures wereentirely resistant to .8AP-induced neurotoxicity and alsodemonstrated no dichlorofluorescein fluorescence or in-creased lipid peroxidation after .AP treatment. Moreover,mature slices exposed to ,(AP in the presence of 25 ,uM EUK-8were significantly protected from ,8AP-induced neurotoxicity.EUK-8 also completely blocked 8AP-induced free radicalaccumulation and lipid peroxidation. These results not onlysupport a role for oxygen free radicals in ,IAP toxicity but alsohighlight the therapeutic potential of synthetic radical scav-engers in Alzheimer disease.

The insoluble extracellular deposits that constitute senileplaques, which are hallmark features of Alzheimer disease(AD), are primarily composed of aggregated ,B-amyloid pro-tein (/3AP) fragments (1). The observation that these plaquesare associated with areas of selective neuronal loss (2), com-bined with recent findings indicating that some forms of ADare related to mutations of the amyloid precursor protein(3-5), has sparked extensive research into the associationbetween I3AP accumulation and neuronal degeneration. Therecent report that transgenic mice overexpressing the humanamyloid precursor protein develop several pathological man-ifestations of AD also strongly supports the involvement of,BAP in AD (6). Although ,BAP has generally been found toproduce neurotoxicity in vitro and in vivo (7, 8), there is littleagreement in the literature concerning the cellular pathways(i.e., apoptosis or necrosis) involved in RAP-induced neuronaldeath (9-11) or the mechanisms by which 1AP exerts its toxiceffect (12, 13). One major issue in studying ,AP-inducedneurotoxicity lies in the choice of experimental models. In vivof3AP injection is not always neurotoxic (14), and most reportsdescribing neuronal ,BAP toxicity utilize dissociated primary

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

neuronal or PC-12 cultures. However, dissociated neuronsretain none of their original connectivity and exist in anonphysiological monolayer, whereas PC-12 cells exhibit onlyselect neuronal properties. In the present study, we used anorganotypic hippocampal culture system to evaluate the neu-rotoxicity of 3AP. Organotypic cultures combine the accessi-bility and maintenance of in vitro culture systems while pre-serving intact the hippocampal synaptic circuitry and anatomy.This culture system has recently been used quite extensively tostudy neuronal death associated with excitotoxicity (15-17),hypoxia and hypoglycemia (18, 19), and knife-cut lesions (20).Although the mechanisms underlying I3AP toxicity are not

yet understood, it has been proposed that reactive oxygenspecies (ROS) play a critical role in producing neuronaldamage (21, 22). This idea was based in part on the protectiveeffect ofROS scavengers against jAP-induced neuronal death(12, 21-25). We previously showed that the synthetic catalyticscavenger of oxygen free radicals, the saleno-manganese com-plex EUK-8, provided a significant degree of protectionagainst hypoxia- and acidosis-induced neuronal damage inhippocampal slices (26). We therefore tested the protectiveeffects of EUK-8 on PAP-induced toxicity in hippocampalslice cultures. Neuronal injury was quantified by determiningthe extent of neuronal uptake of the fluorescent dye propidiumiodide (PI) and of lactate dehydrogenase (LDH) release intothe medium. Additionally, as it has been reported that disso-ciated neurons maintained in cultures for a few days areresistant to ,BAP toxicity (27), we determined the effects of,BAP on hippocampal slices maintained in cultures for a fewdays or a few weeks. The role of ROS in ,BAP-induced injurywas further investigated by determining the levels of freeradical accumulation [as indicated by increased 2',7'-dichlo-rofluorescein (DCF) fluorescence] and lipid peroxidation (asreflected by the assay of thiobarbituric acid reactive sub-stances) in ,BAP-treated slices from both immature and maturecultures.

MATERIALS AND METHODSMaterials. Minimum essential medium and horse serum

were purchased from GIBCO. EUK-8 was provided by Eu-karion (Bedford, MA), and f3AP (1-40 and 1-42) was ob-tained from C. Glabe and prepared as described in Burdick etal. (28) and stored lyophilized until use. 2',7'-Dichlorofluo-rescin diacetate (DCF-DA) was purchased from MolecularProbes. All other chemical products were purchased fromSigma.

Culture Techniques. Organotypic hippocampal cultureswere prepared using the technique of Stoppini et al. (29).Briefly, hippocampi were harvested from 5- to 7-day-old

Abbreviations: ,BAP, ,B-amyloid peptide; AD, Alzheimer disease;LDH, lactate dehydrogenase; PI, propidium iodide; DCF, 2',7'-dichlorofluorescein; DCF-DA, 2',7'-dichlorofluorescin diacetate;ROS, reactive oxygen species; MDA, malondialdehyde.tPresent address: Center on Aging, Sanders Brown Building, Univer-sity of Kentucky Medical Center, Lexington, KY 40536-0230.

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Sprague-Dawley rat pups under sterile conditions and cuttransversely at 400 ,zm using a McIlwain tissue chopper. Tissuesections were then plated onto 30-mm cell culture inserts(Millicell-CM; Millipore; three slices per insert) and main-tained in a humidified CO2 incubator. Culture medium waschanged twice weekly, and sections were allowed to equilibrateto in vitro conditions for at least 5-7 days before treatment.Immature cultures refer to hippocampal slices that weremaintained in vitro for only 7 days, whereas mature culturescorrespond to slices that were kept for 4 weeks before treat-ment.PAP Treatment. ,BAP (1-40 or 1-42) was initially solubi-

lized in sterile H20, incubated overnight at room temperature,and then applied to slice cultures at various concentrations (50,100, or 250 ,ug/ml) in serum-free exposure medium. Similarresults were obtained with either peptide, and unless otherwisenoted, ,BAP in the text refers to either one. A 100-,lI aliquotof ,BAP containing medium was applied directly to the top ofslices at the onset of the exposure to ensure sufficient neuronalexposure to the aggregated peptide (the slices were coveredonly for a few minutes, as the medium rapidly diffused throughthe slices). Selected sections were also treated with EUK-8,which was applied to the medium at the same time as P3AP.Control sections were subjected to the exact same procedurebut did not receive P3AP or EUK-8. At 24, 48, and 72 hr afterexposure onset, the extent of neuronal injury was determinedas described below. To eliminate the possibility that EUK-8directly interfered with the formation of insoluble aggregatesof PAP, we determined the amount of aggregated P3AP afterincubation of P3AP with EUK-8 in H20 for up to 72 hr or inthe medium at the end of the incubation with slices. Aggre-gated ,BAP was collected by centrifugation, and the concen-tration of proteins in the pellet was determined with theBradford assay (30). Under both conditions, EUK-8 was foundnot to reduce the amount of aggregated PAP.Assessment of Neuronal Injury. The extent of neuronal

injury was evaluated at the termination of ,BAP exposure andwas based on two different parameters: (i) the extent ofneuronal uptake of the fluorescent dye PI, and (ii) the amountof LDH released into the medium. PI uptake into damagedneurons was observed with a fluorescent microscope, using astandard rhodamine filter set. Neuronal PI uptake was semi-quantitatively analyzed in sections by assigning a score of 0, 1,2, 3, or 4 to each section corresponding to 0%, 25%, 50%, 75%,or 100% of neurons exhibiting distinct fluorescence. LDHactivity in the medium was measured as described by Koh andChoi (31).

After PI and LDH evaluations, the tissue was removed fromthe membranes and homogenized in ice-cold phosphate-buffered saline. The formation of ROS and the degree of lipidperoxidation were determined by analyzing DCF fluorescenceand malondialdehyde (MDA) release, respectively. DCF-DA(25 ,M) was applied to the culture medium at the onset ofPAP exposure. DCF-DA is freely permeable, and once insidecells it is readily converted to 2',7'-dichlorofluorescin, which isthen able to interact with intracellular free radicals andperoxides to form the highly fluorescent DCF. DCF fluores-cence was detected using a spectrophotofluorimeter withexcitation wavelength set at 502 nm and emission wavelengthset at 523 nm. Standard curves were prepared using knownamounts of DCF, and results were calculated as nanomoles ofDCF formed per milligram of protein and then converted toa percentage of the control. As EUK-8 alone caused a mildincrease in DCF fluorescence even when incubated in theabsence of cells (data not shown), DCF fluorescence in PAP-plus EUK-8-treated sections was expressed as a percentage ofthe value measured in EUK-8 alone treated sections. MDAconcentration was determined by using the thiobarbituricacid-reactive substances assay, as described by Kovachich andMishra (32). Briefly, hippocampal slices were homogenized in

ice-cold phosphate-buffered saline, the homogenates wereprecipitated in 5% trichloroacetic acid, and an aliquot of thetrichloroacetic acid precipitate was incubated with 0.335%thiobarbituric acid in glacial acetic acid for 1 hr at 90-95°C.Lipids were then extracted in butanol, and the optical densitywas measured spectrophotometrically at 540 nm. A standardcurve was established using known amounts of MDA equiv-alents under identical assay conditions, and the results werecalculated as nanomoles of MDA released per milligram ofprotein and then converted to a percentage of the control.

Statistical Analyses. In each experiment, four inserts (i.e.,12 slices) were subjected to the same treatments, and eachexperiment was reproduced two or three times. Therefore dataare the means of at least 24 values (slice data) or 8 values (LDHdata). All data were analyzed using one-way ANOVA, fol-lowed by least significant difference post-hoc analysis todetermine statistical significance. P values <0.05 were consid-ered statistically significant.

RESULTS

.iAP Toxicity in Organotypic Hippocampal Cultures. Theapplication of I3AP (100 ,ug/ml) to mature hippocampal slicecultures resulted in widespread neuronal injury, as indicated byboth PI uptake and LDH release (Fig. 1). After 48 hr ofcontinuous PAP exposure, about 25% of neurons were posi-tively fluorescent, and this value continued to increase to reach

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FIG. 1. Time course of ,BAP-induced PI uptake and LDH releasein the absence or presence of EUK-8. Mature organotypic hippocam-pal cultures were exposed to ,BAP at 100 ,ug/ml for 24, 48, or 72 hr inthe absence or presence of 25 ,uM EUK-8, and the extent of toxicitywas then determined by quantification of PI uptake (A) and LDHrelease (B) as described in Materials and Methods. Data are means ±

SEM of at least 24 (A) or 8 values (B). *, P < 0.05 as compared tocontrol levels (the difference between control values at 0 and 72 hr isnot statistically significant in A, but it is in B).

*- Control *A BAP (100 ggla)

-* BAP+ EUK-8 (25 IM)

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near 40% by 72 hr (Fig. 1A). Hippocampal slices exposed tof3AP in the presence of 25 ,uM EUK-8 were completelyprotected against the f3AP-induced increase in PI uptake (Fig.1A), whereas incubation of cultures with EUK-8 alone had noeffect on PI uptake (data not shown). The semiquantitativeanalysis employed to quantify PI uptake is based on overallfluorescence of the entire slice, but a qualitative evaluation of,AP-induced PI uptake revealed considerably higher fluores-cence in pyramidal neurons as compared to dentate granuleneurons, with no obvious difference in PI uptake betweenCA1, CA3, or hilar neurons (data not shown). f3AP exposurealso resulted in significant increases in LDH release (Fig. 1B),and the increase in LDH release was of the same order ofmagnitude of that previously observed after excitotoxin treat-ment (17). LDH release from ,BAP-treated sections was sta-tistically significantly different from control slices at 72 hr, atwhich time there was a 70% increase in LDH release ascompared to untreated controls (Fig. 1B). As was observedwith PI uptake, slices treated with ,BAP in the presence of 25,uM EUK-8 were completely protected against f3AP-inducedLDH release (Fig. 1B), whereas incubation with EUK-8 alonehad no effect on LDH release (data not shown). Biochemicaland histological analyses indicated that control cultures wereable to tolerate the 72-hr period of serum deprivation with noobvious signs of distress (A.J.B. and M.B., unpublished obser-vations). On the basis of these observations, we assume that the

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steady increase in LDH release in control cultures reflectsaccumulation of LDH in the medium through basal, consti-tutive release mechanisms, rather than a general decline inculture viability.The effects of increasing concentrations of ,BAP on neuronal

PI uptake and LDH release were also determined (Fig. 2).,BAP (72-hr exposure) at 50 ,ug/ml did not significantly affectneuronal PI uptake (Fig. 2A), but ,BAP at 100 and 250 ,ug/mlcaused significant increases in PI uptake into 30% and 45% oftotal neuronal population, respectively (Fig. 2A). Cotreatmentwith 25 ,uM EUK-8 significantly attenuated PI uptake inducedby ,BAP at either 100 to 250 ,ug/ml (Fig. 2A). Similar resultswere obtained with LDH release as increasing ,BAP doses(50-250 jig/ml) produced significant, dose-dependent in-creases (85-150% increase), which were nearly completelyblocked by 25 ,uM EUK-8 (Fig. 2B).

,BAP Toxicity and Oxidative Stress in Immature and Ma-ture Cultures. It has been reported that dissociated neuronsmaintained in vitro for less than 4-5 days are resistant to ,BAPtoxicity (27). We were therefore interested in investigating theeffects of ,BAP application to immature hippocampal slicecultures. Neurons in hippocampal slice cultures have beenshown to develop in a parallel manner to in vivo hippocampalneurons in terms of excitotoxic susceptibility (17), long-termpotentiation (29), and synaptic reorganization after lesion(20). Exposure of immature cultures to ,BAP at 100 ,ug/ml for72 hr had no effect on PI uptake, whereas mature culturestreated with the same 3AP solution showed the characteristicincrease in PI fluorescent neurons (Fig. 3A). EUK-8 treatment

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FIG. 2. Effects of various concentrations of I3AP on PI uptake andLDH release in hippocampal cultures in the absence or presence ofEUK-8. Mature organotypic hippocampal cultures were exposed to,BAP at 50, 100, or 250 ,ug/ml for 72 hr in the presence or absence of25 ,uM EUK-8, and the extent of toxicity was determined by quanti-fication of PI uptake (A) and LDH release (B) as described in Materialsand Methods. Data are means ± SEM of at least 24 (A) or 8 values (B).*, P < 0.05 as compared to I3AP alone.

Imiimature Miature

FIG. 3. Effects of EUK-8 on ,BAP-induced PI uptake and LDHrelease in immature and mature hippocampal slice cultures. Immatureand mature hippocampal slice cultures were exposed to ,BAP at 100,ug/ml for 72 hr in the presence or absence of 25 ,LM EUK-8, and theextent of toxicity was then determined by quantification of PI uptake(A) and LDH release (B) as described in Materials and Methods. Dataare means ± SEM of at least 24 (A) or 8 values (B). *, P < 0.05 ascompared to control levels.

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nearly completely blocked the ,BAP-induced PI uptake inmature cultures, but had no effect on basal PI uptake in eitherimmature or mature cultures (Fig. 3A). Additionally, immaturecultures did not show any increase in LDH release after 72 hrof continuous ,BAP exposure, whereas mature cultures exhib-ited a 75% increase in release, which was significantly atten-uated by 25 ,M EUK-8 (Fig. 3B). Basal LDH release inimmature cultures was somewhat higher than in mature cul-tures, but as there were no other biological or morphologicalindications of neuronal injury, we assume this reflects a greaterlevel of basal release from immature cultures. Indeed, there issome degree of constitutive cell loss during in vitro mainte-nance (33), and, if the LDH values were presented as unit LDHactivity per milligram of protein rather than per milliliter ofmedium, the basal levels of release in immature and maturecultures would be identical.The degree of oxidative stress (ROS accumulation and lipid

peroxidation) after ,BAP exposure was also determined tofurther characterize the role of ROS in ,BAP toxicity. Theaccumulation of oxygen free radicals and hydroperoxides wasestimated by the conversion of dichlorofluorescin to the highlyfluorescent DCF, as described in Materials and Methods.Exposure of hippocampal slices to ,BAP at 100 ,ug/ml for 72 hrcaused DCF fluorescence to significantly increase by 35%above control levels in mature cultures, and this effect wascompletely inhibited by cotreatment with 25 ,uM EUK-8 (Fig.4A). In contrast, immature cultures showed no increase inDCF fluorescence when exposed to the same ,BAP solution

A

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FIG. 4. Effects of EUK-8 on PAP-induced oxidative stress inimmature and mature hippocampal slice cultures. Immature andmature hippocampal slice cultures were exposed to 8AP at 100 ,ug/mlfor 72 hr in the presence or absence of 25 ,M EUK-8, and the extentof oxidative stress was then determined by quantification of DCFfluorescence (A) and MDA release (B) as described in Materials andMethods. Data are means ± SEM of at least eight values. *, P < 0.05as compared to control levels.

(Fig. 4A). The degree of lipid peroxidation produced by I3APtreatment of immature and mature cultures was estimated byassaying thiobarbituric acid-reactive substances (32). Treat-ment of mature cultures with f3AP at 100 ,ug/ml (72-hrexposure) resulted in a significant increase (+26%) in MDAlevels as compared to control values, and this effect wascompletely blocked by 25 ,uM EUK-8 (Fig. 4B). As wasobserved with DCF fluorescence, immature cultures showedno increase in MDA release after ,3AP treatment (Fig. 4B).EUK-8 alone did not alter MDA release from immature ormature cultures (Fig. 4B).

DISCUSSIONThe present study provides strong evidence in support of thehypothesis that ROS mediate P3AP-induced neuronal injuryand indicates that organotypic hippocampal cultures representa model with which to study ,BAP toxicity. This model isespecially appropriate as there is discrepancy between thereported in vitro and in vivo effects of PAP. Although PAPapplication in vitro (to either neurons or neuronal cell lines)has been shown to be neurotoxic (7, 8), application of PAP invivo has produced mixed results in the hands of differentinvestigators (8, 34, 35) and may be toxic only if applied aftertreatment with streptotozotocin or some other glucose-destabilizing agent (36). Using organotypic hippocampal slicecultures, we observed a reliable pattern of neuronal damagewhen P3AP was applied under serum-free medium conditions.This is in contrast to the report that ,BAP toxicity was notobserved unless the peptides (1-40 or 25-35) were microin-jected directly into the neurons (37). Possible reasons for thedifferent results include the method of preparation and main-tenance of the cultured slices (roller-tube vs. membrane cul-tures) and, more importantly, the presence or absence ofserum. It is possible that extended periods of serum depriva-tion stress neurons in such a way as to make them moresusceptible to ,BAP toxicity. However, no discernible biochem-ical or histological indications of significant neuronal injurywere ever present in control cultures, suggesting that neuronswere able to tolerate these conditions.The extent of PI uptake and LDH release has been used

previously by us (17), as well as others (16, 31, 38), to quantifyneuronal injury and death in culture systems. Although thereis generally a good correlation between these two indices, ,BAPexposure had a more robust effect on PI uptake than on LDHrelease in the present study. It is generally accepted that P3APmust be in an aggregated form to exert neurotoxic effects (7).A larger effect of ,BAP treatment on PI uptake as compared toLDH release might therefore reflect the accumulation ofaggregated ,BAP on the superficial layers of the slice, which isthe main region of the slices observed when measuring PIfluorescence (this might also account for the relatively highconcentrations of ,BAP required in our culture system, asaggregated ,BAP is likely to poorly diffuse through the mem-branes and into the cultures). This also suggests that the lackof toxic effect of fAP in immature cultures is not simply dueto the decreased penetration of aggregated PAP as they arethicker than mature cultures, since PI fluorescence shouldhave still been able to detect such an effect. Furthermore, wepreviously reported that immature cultures are in fact moresensitive to the toxic effects of N-methyl-D-aspartate thanmature cultures (17), which excludes the possibility that thedifferential effect of P3AP reflects a generalized difference insensitivity between immature and mature cultures or a differ-ential ability to tolerate serum-free conditions.The measurement of DCF fluorescence as an indicator of

ROS accumulation has previously been used to demonstrateROS production during ,BAP exposure in dissociated neurons(12, 25, 39). The assay of thiobarbituric-reactive substances,which measures MDA released from oxidized lipid bilayers,

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has also been widely used to document lipid peroxidation aftera variety of neuronal insults (26, 40, 41). Although the laterassay has its flaws, the combination of these two assaysprovides an accurate representation of the degree of oxidativestress in neuronal slice cultures after 13AP application. Bothassays clearly indicate that 13AP treatment of hippocampalslices in culture produces oxidative injury. Thus, our results arein good agreement with several studies indicating the involve-ment of free radicals in ,BAP toxicity and the protective effectsof free radical scavengers (12, 25, 39, 42). In our report, wepresent three lines of evidence in support of a pivotal role forROS in ,BAP toxicity: (i) ,BAP-induced PI uptake and LDHrelease were nearly completely blocked by coincubation withEUK-8, a synthetic catalytic free radical scavenger; (ii) 63APapplication to mature cultures resulted in significant increasesin DCF fluorescence and lipid peroxidation, both of whichwere completely inhibited by EUK-8; and (iii) immaturecultures, which were not susceptible to ,AP toxicity, did notshow any increase in DCF fluorescence or lipid peroxidation.EUK-8 has been well characterized in terms of both its freeradical scavenging ability (43) and its effectiveness in in vitromodels of oxidative neuronal injury (26). Furthermore, EUK-8did not affect f3AP aggregation in test tubes or in the sliceculture medium (data not shown). The inhibition of DCFfluorescence and MDA release by EUK-8 also supports itsspecific action as an oxygen free radical and/or peroxidescavenger. The use of immature cultures as a negative controlis a novel approach to explore ,BAP toxicity. Although culturesof this age are also resistant to kainic acid lesioning, they arehighly sensitive to N-methyl-D-aspartate toxicity (17). Thus,their resistance to f3AP-induced toxicity is not a consequenceof some artifact of plating that confers a general, but tempo-rary, resistance to exogenously applied toxins. The demon-stration that ,BAP application to immature cultures does notresult in increased DCF fluorescence and MDA release indi-cates that these parameters are integral to the manifestationsof damage and therefore are not nonspecific consequences ofpeptide aggregation in the presence of neurons. Additionally,the inhibition of the increases in these parameters (in suscep-tible neurons) by the antioxidant EUK-8 indicates that bothDCF fluorescence and MDA release occur downstream to freeradical production. The mechanisms by which 13AP induces theproduction of ROS in susceptible neuronal populations are notilluminated by these studies. It has been proposed that 63APtoxicity is related to the secondary and tertiary structures ofthe peptides (44) and is contingent upon disruption in calciumhomeostasis and subsequent calcium overload (42). The easeof preparation of the hippocampal slice cultures should makeit an interesting model preparation to study the cellular andmolecular mechanisms of f3AP neurotoxicity.

In summary, our results provide a model with which to studyI3AP-induced neuronal injury and highlight some interestingcharacteristics of ,BAP toxicity. The confirmation that ROSplay a critical role in ,BAP toxicity offers further support forclinical developments of antioxidants in the treatment of ADand expend the widening field of oxygen free radical researchin aging. The elucidation of the factors that confer resistanceto f3AP-induced oxidative stress and toxicity in immaturecultures would also have far reaching implications for the studyof the mechanisms of AD susceptibility, as well as for thera-peutic interventions in AD patients.

This work was supported by a grant from Eukarion, Inc. (Bedford,MA).

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