Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures

Brain Research 917 (2001) 21–44 www.elsevier.com / locate / bres

Research report

Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures Bjarne W. Kristensen a

a,b ,

*, Jens Noraberg a,b , Jens Zimmer a,b

Anatomy and Neurobiology, Inst. of Medical Biology, SDU-Odense University, Winsløwparken 21, DK-5000, Odense C, Denmark b NeuroScreen ApS, Inst. of Medical Biology, SDU-Odense University, Winsløwparken 21, DK-5000 Odense C, Denmark Accepted 27 July 2001

Abstract The excitotoxic profiles of (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA), (RS)-2-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartate (NMDA) were evaluated using cellular uptake of propidium iodide (PI) as a measure for induced, concentration-dependent neuronal damage in hippocampal slice cultures. ATPA is in low concentrations a new selective agonist of the glutamate receptor subunit GluR5 confined to KA receptors and also in high concentrations an AMPA receptor agonist. The following rank order of estimated EC 50 values was found after 2 days of exposure: AMPA (3.7 mM).NMDA (11 mM)5KA (13 mM).ATPA (33 mM). Exposed to 30 mM ATPA, 3 mM AMPA and 10 mM NMDA, CA1 was the most susceptible subfield followed by fascia dentata and CA3. Using 8 mM KA, CA3 was the most susceptible subfield, followed by fascia dentata and CA1. In 100 mM concentrations, all four agonists induced the same, maximal PI uptake in all hippocampal subfields, corresponding to total neuronal degeneration. Using glutamate receptor antagonists, like GYKI 52466, NBQX and MK-801, inhibition data revealed that AMPA excitotoxicity was mediated primarily via AMPA receptors. Similar results were found for a high concentration of ATPA (30 mM). In low GluR5 selective concentrations (0.3–3 mM), ATPA did not induce an increase in PI uptake or a reduction in glutamic acid decarboxylase (GAD) activity of hippocampal interneurons. For KA, the excitotoxicity appeared to be mediated via both KA and AMPA receptors. NMDA receptors were not involved in AMPA-, ATPA- and KA-induced excitotoxicity, nor did NMDA-induced excitotoxicity require activation of AMPA and KA receptors. We conclude that hippocampal slice cultures constitute a feasible test system for evaluation of excitotoxic effects and mechanisms of new (ATPA) and classic (AMPA, KA and NMDA) glutamate receptor agonists. Comparison of concentration–response curves with calculation of EC 50 values for glutamate receptor agonists are possible, as well as comparison of inhibition data for glutamate receptor antagonists. The observation that the slice cultures respond with more in vivo-like patterns of excitotoxicity than primary neuronal cultures, suggests that slice cultures are the best model of choice for a number of glutamate agonist and antagonist studies.  2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Excitatory amino acids: excitotoxicity Keywords: Propidium iodide; MAP2; NeuN; Neurodegeneration; Neuroprotection; NBQX; GYKI 52466; MK-801

1. Introduction The principal excitatory transmitter in the central nervous system, L-glutamate, acts through three classes of ionotropic receptors, named after the respective and now classic glutamate receptor agonists 2-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartate (NMDA). The excitotoxic *Corresponding author. Tel.: 145-6550-3800; fax: 145-6590-6321. E-mail address: [email protected] (B.W. Kristensen).

effects of glutamate and its agonists have been studied in various experimental in vivo [54,68,92] and in vitro [39] models. Together with glutamate receptor antagonists, they have provided evidence that activation of glutamate receptors plays a key role in various neurodegenerative diseases and conditions [13,18]. ATPA ((RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol4-yl)propionic acid) is a ‘new’ KA receptor agonist originally introduced as an AMPA receptor agonist [53]. It has recently gained renewed interest as a very selective agonist for the KA receptor subunit GluR5, when applied

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02900-6

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in low concentrations [15]. Based on this property and its ability to cross the blood–brain barrier allowing systemic administration in animals [1,99], ATPA is currently being studied both in vivo [86] and in vitro [16,17,61,97]. Organotypic hippocampal slice cultures have previously been used to study KA- and NMDA-induced neurodegeneration in different experimental situations, but the involvement of AMPA, KA and NMDA receptors in experimental neuronal degeneration is not known [6,7,9,29,87,102]. The details of hippocampal subfield vulnerability to AMPA are not known either, but clearly of importance. It has thus been demonstrated that AMPA receptors, and not NMDA receptors, are involved in delayed ischemic neuronal cell death, which should be accessible for therapeutical intervention [21,45,111]. Having developed standardized protocols including quantifiable markers for neurodegeneration [74], we therefore characterized the excitoxic effects of ATPA in comparison with AMPA, KA and NMDA on hippocampal CA1 and CA3 pyramidal cells and dentate granule cells. Using cellular uptake of the fluorescent dye propidium iodide (PI) as a quantifiable marker for neuronal degeneration [74,102], we were able to estimate and compare both the concentrations of ATPA, AMPA, KA and NMDA that induced half-maximal PI uptake (EC 50 value) and the susceptibility of the individual hippocampal subfields to each of these compounds, when applied in concentrations close to their EC 50 values. ATPA sensitive GluR5 receptors are expressed both by CA3 pyramidal cells [100] and GABAergic CA1 interneurons [78]. It might therefore be expected that a KA receptor stimulating component of ATPA would present itself with an excitotoxic profile different from AMPA and KA, since AMPA and KA receptors have a different cellular distribution [2,36,46,72]. The possible interactions between the different receptors after application of ATPA, AMPA, KA and NMDA were investigated by co-application studies using the competitive AMPA / KA receptor antagonist 2,3-dihydroxy-6-nitro7-sulfamoyl-benzo(F)-quinoxaline (NBQX) [92], the noncompetitive AMPA / KA receptor antagonist 2,3-benzodiazepine GYKI 52466 [22] and the non-competitive NMDA receptor antagonist (1)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine (MK-801) [110]. Bringing all the results together moreover helped to demonstrate that hippocampal slice cultures display more in vivo-like responses than reported for primary cultures of hippocampal (and cortical) neurons. Preliminary reports of this study have appeared in abstract form [49,50].

2. Materials and methods

2.1. Organotypic slice cultures Hippocampal slice cultures were prepared and grown

according to the interface culture method [98], modified by Noraberg et al. [51,74]. In brief, 5–7-day-old rats of Wistar strain (Møllegaard, Denmark) were killed by instant decapitation, the two hippocampi isolated and their dorsal halves cut in transverse sections at 300–350 mm by a McIlwain tissue chopper. The tissue slices were inspected, separated and trimmed for excess tissue under a microscope, and then in random order placed on porous (0.4 mm), transparent insert membranes (30 mm in diameter) (Millipore Corp., Bedford, MA, USA, Cat. No. PICM 030 50) with six slices on each membrane. The inserts were transferred to 6 well culture trays (Corning Costar, Corning, NY, USA), where each well contained 1 ml culture medium, composed of 50% Opti-MEM (Cat. No. 31985047), 25% horse serum (Cat. No. 26050-047), 25% Hank’s BSS (HBSS; Cat. No. 24020-091) (all from Gibco BRL), supplemented by D-glucose to a final concentration of 25 mM. The trays were kept in an incubator with 5% CO 2 and 95% atmospheric air at 368C. After 4 days of incubation the culture medium was replaced with 1 ml of chemically defined, serum-free Neurobasal medium (Gibco BRL, Cat. No. 21103-049) with 25 mM D-glucose and 1 mM Lglutamine (Sigma, Vallensbæk Strand, Denmark, Cat. No. 25030-024), and 2% B27 supplement (GIBCO BRL, Cat. No. 17504-010) [8]. The medium was thereafter changed twice a week for the next 3–4 weeks. No antimitotic drugs or antibiotics were used at any stage. The cultures were regularly checked by low magnification and light and phase contrast microscopy to ensure that sufficient numbers of cultures with intact structures were available and allocated to the different control and experimental series.

2.2. Exposure to glutamate receptor agonists and antagonists Neurotoxic effects were primarily monitored by PI uptake, recorded densitometrically for the entire culture and for the individual subfields (see below). For determination of half-maximal excitotoxic concentrations (EC 50 values) 3–4-week-old slice cultures were exposed for 48 h to 0.1–300 mM ATPA (a generous gift from Prof. Povl Krogsgaard-Larsen, Copenhagen, Denmark), 0.1–100 mM AMPA (Sigma, Cat. No. A-6816), 0.1–300 mM KA (Sigma, Cat. No. K-025) or 0.3–300 mM NMDA (Sigma, Cat. No. M-3262), and the PI uptake recorded for the entire culture (see below). The vulnerability of the individual hippocampal subfields to ATPA, AMPA, KA and NMDA was determined by exposing different groups of cultures, prepared at the same time, to one of the four compounds in concentrations close to the respective EC 50 values. In order to express the resulting damage (PI uptake) in percentage of maximal neuronal death (maximal PI uptake), the cultures were thereafter exposed to 50 mM glutamate. To ensure that the maximal PI uptake induced by the

B.W. Kristensen et al. / Brain Research 917 (2001) 21 – 44

high concentrations of each agonist was the same and thereby comparable, we exposed groups of cultures from the same batch to 100 mM of ATPA, AMPA, KA or NMDA, which for all, according to the individual agonist concentration–response curves, would induce maximal PI uptake. In order to check that the maximal PI uptake actually corresponded to total neuronal death, cryostat sections of the cultures were stained with toluidine blue for general cell staining and immunocytochemical staining for the neuronal markers neuron specific protein (NeuN) and microtubule-associated protein 2 (MAP2) (see below). The involvement of AMPA / KA receptors in ATPA-, AMPA-, KA- and NMDA-induced degeneration was studied by exposing groups of cultures from the same batch to 30 mM ATPA, 3 mM AMPA, 8 mM KA or 10 mM NMDA together with 1–20 mM concentrations of the competitive AMPA / KA receptor antagonist NBQX (generous gift from Novo Nordisk A / S, Bagsværd, Denmark). Other cultures were exposed to 30 mM ATPA, 3 mM AMPA or 8 mM KA together with 10–300 mM concentrations of the non-competitive AMPA / KA receptor antagonist GYKI 52466 (generous gift from Prof. F. Nicoletti, Catania, Italy). The involvement of NMDA receptors in the mechanisms of ATPA-, AMPA-, KA- and NMDA-induced degeneration was investigated by exposing cultures to 30 mM ATPA, 3 mM AMPA, 8 mM KA or 10 mM NMDA together with 0.3–10 mM concentrations of the non-competitive NMDA receptor antagonist MK-801 (RBI, Cat. No. M-107). The medium was changed to medium with glutamate receptor antagonists 30 min before addition of glutamate receptor agonists. The experimental series always included control cultures not subjected to glutamate receptor agonist or antagonist treatment.

2.3. Propidium iodide uptake Propidium iodide (PI, 3,8-diamino-5-(3-(diethylmethylamino)propyl)-6-phenyl phenanthridinium diiodide; Sigma, Cat. No. P4170) is a very stable flourescent dye absorbing blue–green light (493 nm) and emitting red fluorescence (630 nm). As a polar compound it only enters dead or dying cells with a damaged or leaky cell membrane. Once inside the cell PI interacts with DNA to yield a bright red fluorescence. PI is basically nontoxic to neurons [35,83], and has due to the above mentioned properties been used as an indicator of neuronal membrane integrity [101] and cell damage [44,83,84,102]. In the present study PI was used to quantify neuronal degeneration in accordance with a previously developed protocol [51,74]. After 3–4 weeks of culturing, 20 ml of 0.1 mM PI was added to the culture medium to achieve a final concentration of 2 mM PI. This concentration was used in the medium at all subsequent medium changes, including the media used to expose the cultures to gluta-

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mate receptor agonists and antagonists. In order to monitor the basic levels of PI uptake and cell death in the cultures, PI was added 3–12 h before the exposures to glutamate receptor agonists and the PI uptake at this and subsequent stages recorded by fluorescence microscopy (Olympus IMT-2, 43(Splan FL2)), using a standard rhodamine filter and a digital camera (Sensys KAF 1400 G2, Photometrics, Tucson, AZ, USA). After digital recording of the basic PI uptake, the cultures were exposed to either ATPA, AMPA, KA or NMDA alone or in combination with NBQX, GYKI 52466 or MK-801. The PI uptake in the individual cultures was recorded again after 24 h (Day 1) and 48 h (Day 2). In order to compare of the relative susceptibility of the individual hippocampal subfields to ATPA, AMPA, KA and NMDA, all neurons were killed by exposure to 50 mM of glutamate for 1 h followed by 24 h in normal medium (with PI), before a final set of pictures representing maximal PI uptake was taken [51,73,74]. In this way the PI uptake induced by ATPA, AMPA, KA or NMDA and recorded at Days 1 and 2 could be expressed in percentage of the maximal PI uptake (see above). In all other experiments the PI uptake was expressed in arbitrary units and the treatment with 50 mM glutamate followed by recording of PI uptake used to illustrate the cytoarchitecture of the culture and its subfields. The digital photos were analyzed by densitometry in NIH Image 1.62 image analysis program (National Institute of Health, USA) after outlining the neuronal layers.

2.4. Determination of GAD activity Glutamic acid decarboxylase (GAD) activity was assessed in tissue samples pooled from three hippocampal cultures taken from the same culture well. The tissue was sonicated and the GAD activity assessed on the same day. Briefly, 25 ml aliquots of the homogenate and 25 ml of a cocktail of 10 ml L-[1- 14 C]-glutamate (200.000 cpm, 32.5 mM sodium L-[1- 14 C]-glutamate, Amersham, Little Chalfold, UK), 10 ml mercaptoethanol buffer (1:20 v / v 1 M mercaptoethanol in 0.5 mM phosphate buffer with pH 6.9 and containing 2 mg / ml disodium EDTA), and 5 ml pyridoxal phosphate (2.6 mg / 10 ml distilled water), were mixed in a polystyrene conical centrifuge tube and incubated 1 h at 378C. Plastic scintillation vials containing filter paper (Whatmann International Ltd., Kent, UK) with hyamine hydroxide to trap the 14 CO 2 produced by decarboxylation were placed on top of the reaction tubes to which they were connected with latex tubing. After incubation the reaction was stopped by injection of 200 ml 30% trichloroacetic acid through the latex tubing. The next day 4 ml scintillation solution (Ecoscint A, National Diagnostics, Atlanta, Georgia, USA) was added and after 24 h in the dark, the samples were counted in a liquid scintillation counter. GAD activity was expressed as nmol glutamate (Glu) / mg protein / h at 378C.

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2.5. Immunocytochemical staining for NeuN and MAP2 For NeuN and MAP2 immunocytochemical staining, slice cultures were fixed for 1 h in 4% phosphate buffered paraformaldehyde and thereafter transferred to 0.15 M phosphate buffer with 20% sucrose for 24 h for cryoprotection. The individual cultures were then embedded in Cryo-embed (AX-LAB, Copenhagen, Denmark), frozen and cut by cryostat in three series at 20 mm. The slide-mounted sections were subsequently stored at 2208C until the three series were stained by toluidine blue [74] and NeuN- [75] and MAP2-immunostaining [37] according to presently established protocols. Omission of the primary antibody abolished all staining reaction. The NeuN- and MAP2-immunostaining was quantified densitometrically in sections from the middle to upper part of the cultures. After digital photography at a primary magnification of 403, the pyramidal and granule cell layers (NeuN) and the neuropil layers, i.e. stratum radiatum of CA1 and CA3 and the dentate molecular layer (MAP2) were outlined in NIH-Image (see above) and the optical densities recorded.

2.6. Statistics and calculation of EC50 values All densitometric data were expressed as means6standard error of mean (S.E.M.). For illustration of concentration–response data sigmoid shaped curves were fitted to the different concentration–response data sets using Microcal Origin 3.5 (Microcal Software Inc., Northhampton, MA, USA). From these curves the EC 50 values, corresponding to 50% of the plateau level (maximal damage), were obtained. The curves were statistically compared by a Wald test, based on robust variance estimates using Intercooled Stata 6 (Stata Corporation, College Station, TX, USA). For other data, statistical significance was assessed in GraphPad Instat (GraphPad Software, San Diego, CA, USA), using single factor analysis of variance (ANOVA) with Bonferroni correction for comparison of the groups of interest. Inhibition data for glutamate receptor antagonists were compared in Stata by two factor analysis of variance (ANOVA) including interactions in the model. Differences were considered significant at P,0.05.

3. Results

3.1. Dose dependent excitotoxic effects of ATPA, AMPA, KA and NMDA The PI uptake in hippocampal slice cultures before agonist exposure and in unexposed control cultures was very low. Addition of ATPA, AMPA, KA or NMDA to the culture medium resulted in a clear, concentration-dependent increase in PI uptake as recorded 1 and 2 days later

(Fig. 1). The concentration–response curve for AMPA in Fig. 1 is reproduced with permission from [113]. For both Days 1 and 2, the concentration–response curves differed significantly between the glutamate receptor agonists (P, 0.0001 for ATPA versus AMPA, ATPA versus KA, ATPA versus NMDA, AMPA versus KA, and AMPA versus NMDA, and P,0.02 for KA versus NMDA). The EC 50 values, estimated from the sigmoid curve fits for PI uptake in entire hippocampal slice cultures, gave the following rank orders of potency after Day 1: AMPA (4.7 mM). NMDA (12 mM).KA (35 mM).ATPA (45 mM), and after Day 2: AMPA (3.7 mM).NMDA (11 mM)5KA (13 mM).ATPA (33 mM) (Fig. 1 and Table 1). Interestingly, KA had not the same steep concentration–response curves as ATPA, AMPA and NMDA. For all compounds the induced PI uptake was increased at Day 2 compared to Day 1 (P,0.0001). The NMDA-induced PI uptake after Day 1 was significantly higher than for the other glutamate receptor agonists (statistical comparison was made for 100 mM concentrations, P,0.001 for NMDA versus ATPA and KA, and P,0.05 for NMDA versus AMPA). The possible excitotoxic effect of ATPA in low (1 or 3 mM) GluR5 selective concentrations [15] was investigated by a densitometric subfield analysis of the pyramidal cell layer, the strata oriens and radiatum of CA1 and CA3 and of the granule cell layer, but no increase in PI uptake was detected in any of the hippocampal subfields at these concentrations (Fig. 2A–C). The GAD activity measurements appeared slightly, but not significantly reduced for cultures exposed to 1–10 mM ATPA contrasting the total loss of GAD activity after exposure to 30 mM ATPA (EC 50 516.4 mM) (Fig. 2D). To validate the GAD assay, the GAD activity was measured at different concentrations of protein for control cultures and cultures exposed to 3 mM ATPA, but it did not reveal a significant reduction in GAD activity for cultures treated with 3 mM ATPA (data not shown).

3.2. Differential susceptibility of hippocampal subfields to ATPA, AMPA, KA and NMDA The susceptibility of the different hippocampal subfields to ATPA, AMPA, KA and NMDA was analyzed by applying agonist concentrations close to the EC 50 values, based on sigmoid curve fits for PI uptake at Day 2 in entire hippocampal slice cultures. Exposed to 30 mM ATPA, 3 mM AMPA or 10 mM NMDA for 2 days, the PI uptake in the CA1 pyramidal cell layer was significantly higher than the uptake in CA3, with the uptake in the dentate granule cell layer in between (Fig. 3A, B and D and Fig. 4A, B and D; Table 1). The subfield vulnerability data for NMDA used in Fig. 3 is reproduced with permission from [113]. When exposed to 8 mM KA the CA3 pyramidal cell layer was the most susceptible, followed by the dentate granule cell layer and the CA1 pyramidal cell layer (Figs. 3C and 4C; Table 1).

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Fig. 1. Concentration–response relationships between increasing concentrations of ATPA (A), AMPA (B), KA (C) and NMDA (D) and cellular propidium iodide (PI) uptake, measured for entire hippocampal slice cultures (all subfields together) after 24 h (Day 1) and 48 h (Day 2) of exposure. The maximal PI uptake at the plateau-level 2 days after start of exposure is set to 100%. After 2 days of exposure the calculated EC 50 values gave the following rank order of excitotoxic potency: AMPA (3.7 mM).NMDA (11 mM)$KA (13 mM).ATPA (33 mM) (see also Table 1). Note the difference between the concentration–response curves for KA and the other agonists and that the most of the PI uptake for NMDA has occurred already at Day 1, unlike the other agonists. Data are shown as means6S.E.M., with n58–24, **P,0.01, ***P,0.001 using ANOVA with Bonferroni correction for comparison with control. The four curves for Day 1 and Day 2, respectively, were significantly different (P,0.0001) as were the curves for Days 1 and 2 for each agonist (P,0.0001). The curves were compared by a Wald test. For further statistical comparison see Results section. The concentration–response curve for AMPA is reproduced with permission from [113].

3.3. Correspondence between maximal PI uptake, Nissl staining and NeuN- and MAP2 -immunostaining The use of EC 50 values for comparison of excitotoxic potencies requires that the maximal PI uptake induced by ATPA, AMPA, KA and NMDA is similar. In order to verify this, sets of cultures from the same batch were

exposed to 100 mM concentrations of ATPA, AMPA, KA or NMDA and the induced PI uptakes recorded after 2 days for the CA1 and CA3 pyramidal cell layers and the dentate granule cell layer as well as the entire hippocampal cultures. Recorded in this way the PI uptake was similar for the different agonists within the same subregion (Figs. 5 and 6). The PI uptake, was, however different for the

Table 1 Hippocampal slice culture and subfield vulnerability to ATPA, AMPA, KA and NMDA

ATPA AMPA KA NMDA

EC 50 (mM) cultures

entire

Day 1

Day 2

45 4.7 35 12

33 3.7 13 11

% PI uptake in hippocampal subfields

ATPA (30 mM) AMPA (3 mM) KA (8 mM) NMDA (10 mM)

FD

CA3

44.463.4 38.765.1 33.162.6 47.763.7

37.06 28.06 42.76 36.36

CA1 2.7 5.2 2.4 [ 3.3 [[

53.963.1*** 45.564.4* 21.161.9*** , 65.166.1*** ,

[[[ [[[

EC 50 values for Days 1 and 2 were estimated from significantly different concentration–response PI uptake curves obtained after 1 and 2 days of exposure, respectively. The four curves for Day 1 and Day 2, respectively, were significantly different (P,0.0001) as were the curves for Days 1 and 2 for each agonist (P,0.0001). The curves were compared by a Wald test. For further statistical comparison see Results section. Subfield vulnerability data are shown as means6S.E.M., with n517–29 for ATPA, AMPA and KA and n59 for NMDA. *P,0.05, ***P,0.001 (* indicates CA1 versus CA3), [ P,0.05, [[ P,0.01, [[[ P,0.001 ( [ indicates CA1 or CA3 versus FD), ANOVA with Bonferroni correction for comparison of the different subfields (see also Figs. 1, 3 and 4).

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Fig. 2. No distinct cellular uptake of propidium iodide (PI) uptake in control culture, used here to outline hippocampal subfields and layers (A), or in culture exposed to 3 mM ATPA for 48 h (B), indicating that ATPA is without excitotoxic effects at low (1–3 mM) GluR5 selective concentrations. Densitometric measurements of PI uptake confirmed that there was no increase in PI uptake in the CA1, CA3 and fascia dentata cell and neuropil layers in cultures exposed to 1 and 3 mM ATPA (C). GAD activity measurements (glutamate / mg protein / h) of cultures exposed for 48 h to increasing concentrations of ATPA (D) showed no significant decrease in GAD activity after exposure to 1–10 mM ATPA, but 30 mM ATPA abolished all GAD activity (EC 50 516.4 mM ATPA). Data are shown as means1S.E.M. with n512 for the PI uptake and n54–8 for the GAD-activity measurements, ***P,0.001, ANOVA with Bonferroni correction for comparison with control. Abbreviations: FD, fascia dentata; g, granule cell layer; p, pyramidal cell layer; o, stratum oriens; r, stratum radiatum.

different subregions. The ATPA- and AMPA-induced maximal PI uptake was significantly higher in CA1 than in the dentate granule cell layer (P,0.001) and in CA3 (P,0.01) as well as in the entire culture (P,0.001). For KA and NMDA the maximal PI uptake was significantly higher in CA1 than in the dentate granule cell layer (P,0.001) and the entire culture (P,0.001), just as the KA- and NMDA-induced maximal PI uptake in the CA3 was significantly higher than in the dentate granule cell layer (P,0.05 and P,0.001, respectively) (Fig. 6). The correspondence between maximal PI uptake and total neuronal cell death was addressed by ordinary cell

staining and NeuN- and MAP2-immunostaining of the exposed cultures. In toluidine blue stained sections of 4-week-old hippocampal control slice cultures and cultures exposed to 100 mM concentrations of ATPA, AMPA, KA or NMDA, all four glutamate receptor agonists were found to have induced extensive degenerative changes of dentate granule cells and CA1 and CA3 pyramidal cells (Fig. 7). At low magnification the otherwise distinct staining of cell layers, present in control cultures (Fig. 7A), had disappeared (Fig. 7E, I, M, Q). At high magnification the cytoplasm was lighter (arrows, Fig. 7K, L, T) or almost absent (arrows, Fig. 7G, H, P) and the neuronal nuclei

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Fig. 3. Propidium iodide (PI) uptake in hippocampal slice cultures recorded 48 h after start of exposure to 30 mM ATPA (A), 3 mM AMPA (B), 8 mM KA (C) or 10 mM NMDA (D). Note that the regional PI uptake is very similar for AMPA, ATPA and NMDA with CA1 pyramidal cells being most susceptible, while CA3 pyramidal cells are most susceptible to KA.

stained more densely with a small rounded necrotic-like appearance (arrows, Fig. 7). In NeuN immunostaining all hippocampal subfields showed reduced staining after 2 days of exposure (Figs. 8 and 10A) with a change of the individual neurons from having a darkly stained cytoplasm and a lightly stained nucleus to having a weakly stained cytoplasm and a darkly stained nucleus (arrows, Fig. 8). In MAP2 immunostaining there was an almost total loss of MAP2-immunoreactive dendrites in all hippocampal subfields after 2 days of exposure to each of the four agonists (Fig. 9), as verified by densitometric measurements (Fig. 10B). For all agonists, the reduction was most prominent in stratum radiatum of CA1. With 100 mM KA there was an almost total loss in all subfields, whereas

exposure to 100 mM AMPA caused the least reduction. After exposure to 100 mM AMPA 45% of the MAP2 immunoreactivity in the str. radiatum of CA3 and 35% of the dentate molecular layer was preserved, compared to only 5% in the str. radiatum of CA3 and 10% in the dentate molecular layer after 100 mM KA and 15% in the two layers after 100 mM NMDA. At high magnification a few dendrites which appeared to undergo degeneration were found in both CA1, CA3 and fascia dentata after exposure to the different agonists (arrows, Fig. 9).

3.4. Neuroprotective effects of NBQX, GYKI 52466 and MK-801 The involvement of the different receptor types in the

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Fig. 4. Densitometric measurements of propidium iodide (PI) uptake in CA1 and CA3 pyramidal cells and granule cells of fascia dentata (FD). The PI uptake recorded 48 h after start of exposure to 30 mM ATPA (A), 3 mM AMPA (B), 8 mM KA (C) or 10 mM NMDA (D) is expressed as percentage of maximal uptake after exposure to 50 mM glutamate (total neuronal death). Note similar profiles of PI uptake for AMPA, ATPA and NMDA with CA1 being most susceptible, while CA3 is most susceptible to KA. The susceptibility of the dentate granule cells (FD) is intermediate to the susceptibility of the CA1 and CA3 pyramidal cells for all agonists. Data are shown as means1S.E.M. with n517–29 for ATPA, AMPA and KA and n59 for NMDA, *P,0.05, **P,0.01, ***P,0.001, ANOVA with Bonferroni correction for comparison of the different subfields. The subfield vulnerability for NMDA is reproduced with permission from [113].

excitotoxic effects of ATPA, AMPA, KA and NMDA was investigated by applying the competitive AMPA / KA receptor antagonist NBQX, the non-competitive AMPA / KA receptor antagonist GYKI 52466 and the non-competitive NMDA receptor antagonist MK-801. Comparing the four different inhibition curves for NBQX in each subfield, NBQX inhibited AMPA-, ATPAand KA-induced excitotoxicity, whereas NMDA-induced excitotoxicity was insensitive to NBQX (Table 2). The potency of NBQX was highest against AMPA-induced excitotoxicity followed by ATPA and KA-induced excitotoxicity (AMPA versus ATPA, P,0.05 in all subfields; ATPA versus KA, P,0.001 in CA1 and CA3 and P,0.01 in FD; KA versus NMDA, P,0.001 in CA1, CA3 and P,0.01 in FD). Comparing the protection in the different subfields for a given agonist there was in general no significant differences except NBQX being more protective against ATPA in CA1 (P,0.001) and CA3 (P,0.05) compared with fascia dentata. However, 1 mM NBQX

added together with 8 mM KA gave a significant protection (50%) of CA1 pyramidal cells, whereas 10 mM NBQX was necessary for protection of CA3 pyramidal cells (50%) and 20 mM NBQX for protection of dentate granule cells (20%). In 30–300-mM concentrations, GYKI 52466 protected against 30 mM ATPA, 3 mM AMPA and 8 mM KA in all hippocampal subfields. The inhibition data were the same for a given agonist in the different subfields and for the three different agonists within the same subfield (Table 3). Already at 0.3 mM, MK-801 protected completely against 10 mM NMDA-induced damage in all hippocampal subfields (Table 4), whereas a concentration of 10 mM MK-801 was unable to protect any subfield against 30 mM ATPA, 3 mM AMPA and 8 mM KA. Comparison of inhibition data gave the following results: NMDA versus ATPA, P,0.001 in CA1 and CA3 and P,0.01 in FD; NMDA versus AMPA, P,0.001 in CA1, CA3 and FD and NMDA versus KA, P,0.001 in CA1 and CA3 and P,

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Fig. 5. Propidium iodide (PI) uptake in hippocampal slice cultures recorded 48 h after start of exposure to 100 mM ATPA (A), 100 mM AMPA (B), 100 mM KA (C) or 100 mM NMDA (D). Note high and similar PI uptake in all hippocampal subfields after exposure to each of the four agonists at these concentrations.

0.05 in FD. There was no difference between the inhibition data in the three subfields for a given agonist.

4. Discussion Hippocampal slice cultures were used to establish and compare the excitotoxic profiles of ATPA, AMPA, KA and NMDA in terms of EC 50 values and regional differences in vulnerability. Regarding ATPA, our expectation was that the GluR5 receptor stimulating property would present itself in terms of a subfield vulnerability different from AMPA and KA. From previous studies [6,20,51,102], we also expected that the excitotoxic data on ATPA, AMPA, KA and NMDA obtained in the hippocampal slice cultures

would be more in vivo-like than data obtained from primary neuronal cultures [12,39,52,57]. Our expectations relate to the fact that hippocampal slice cultures display a distinct organotypic organization and basically normal distribution of the neuronal cell layers and intrinsic axonal projections, like the dentate CA3mossy fiber and the CA3 to CA1 Schaffer collateral projections, just as the connective reorganization induced by explantation and isolation from the brain follows the principles of in situ lesion studies [112]. Compared with the in vivo situation there is direct access to the brain tissue with no blood–brain barrier and complicated pharmacodynamic and pharmacokinetic issues in slice cultures. The pharmacokinetics in slice cultures may on the other hand not be as simple as in primary cultures with a

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Fig. 6. Densitometric measurements of propidium iodide (PI) uptake in CA1 and CA3 pyramidal cells, dentate granule cells (FD) and entire slice cultures 48 h after start of exposure to 100 mM ATPA, 100 mM AMPA, 100 mM KA or 100 mM NMDA. At this concentration all four agonists induced corresponding, maximal levels of PI uptake in the different subfields and for the entire culture. The densities of maximal PI uptake were highest in CA1, followed by CA3 and the dentate granule cells. Considering that all neurons in the subfields are dead or damaged this most likely reflects differences in cell densities. For ATPA and AMPA the PI uptake in CA1 was significantly higher than for FD (P,0.001) and CA3 (P,0.01) and the entire culture (P,0.001). For KA and NMDA the PI uptake in CA1 was significantly higher than for FD (P,0.001) and the entire culture (P,0.001) and the PI uptake in CA3 was significantly higher than for FD (P,0.05 and P,0.001, respectively). Data are shown as means6S.E.M. with n524, ANOVA with Bonferroni correction for comparison of the different groups.

monolayer cell distribution. Another issue to be kept in mind is that slice cultures usually are made from 1-weekold rat pups and left to develop and mature for 2–4 weeks before exposure to excitotoxins [74,83,84,87,112,113], whereas primary cultures are prepared from late fetal rats and usually kept 1–3 only weeks before exposure [57,76]. The developmental stage of slice cultures is accordingly closer to that of adult or young adult brains used in most in vivo studies [54,64,68,92]. The density, extent and distribution of the induced neuronal degeneration were evaluated by standardized quantitative analysis of the cellular uptake of PI, a compound which has been used increasingly over the last decade as a quantifiable marker for neuronal degeneration in hippocampal slice cultures exposed to e.g. excitotoxins [6,95,102], hypoxia and hypoglycemia alone or in combination [82,85,103].

4.1. Comparison of concentration–response curves and EC50 values By estimating EC 50 values from the PI uptake concentration–response curves (Table 1), we found that the EC 50 value of ATPA after 2 days of exposure was almost one order of magnitude higher than the corresponding EC 50 value for AMPA. This is in accordance with rat cortical wedge electrophysiology [97] and binding experiments [53]. When the AMPA responsive glutamate receptor subunits GluR1, GluR2 and GluR3 were expressed in Xenopus oocytes and exposed to ATPA, the respective electrophysiological EC 50 values were 62, 19 and 26 mM [97]. This overlaps with the excitotoxic EC 50 value of 33

mM ATPA obtained from hippocampal slice cultures in the present study and accordingly suggests that the excitotoxic effect of ATPA is mediated primarily via AMPA receptors. Aiming at specific activation of GluR5 receptors by exposing slice cultures for 2 days to low concentrations of 0.3–3 mM ATPA [15], did correspondingly not result in increased PI uptake or reduced GAD activity. However, an electrophysiological EC 50 value of 2.1 mM ATPA has been observed for homomeric GluR5 receptors expressed in human embryonic kidney (HEK) 293 cells and a value of 0.62 mM ATPA for rat dorsal root ganglion (DRG) neurons [15], thereby making it plausible that this receptor could participate in the excitotoxic effects of ATPA. Recently, the GluR5 receptor subunit has been shown to exhibit novel functional properties by forming heteromeric receptors with the GluR6 receptor subunit, including reduced desensitization and enhanced magnitude of responses to ATPA in comparison with homomeric GluR5 receptors [17]. A natural occurrence of such combinations, which possibly are expressed both by CA3 pyramidal cells [100] and GABAergic CA1 interneurons [78] would accordingly enhance the probability for involvement of GluR5 containing receptors in ATPA excitotoxicity. The excitotoxic potencies of the classic glutamate receptor agonists KA, AMPA and NMDA have previously been compared in different in vivo [54] and in vitro studies [9,28,52,61,94,102], but all three agonists have not been compared in hippocampal slice cultures. By exposing hippocampal slice cultures to selected concentrations of KA and NMDA for 30 min [102] or 3 h [9], earlier studies found that KA was less potent than NMDA. In the present study KA was only slightly less potent than NMDA, when

B.W. Kristensen et al. / Brain Research 917 (2001) 21 – 44

31

Fig. 7. Toluidine blue (Nissl) stained sections of 4-week-old control hippocampal slice culture (A–D) and cultures exposed for 48 h to 100 mM ATPA (E–H), 100 mM AMPA (I–L), 100 mM KA (M–P) or 100 mM NMDA (Q–T). All four glutamate receptor agonists induced distinct degenerative changes in both dentate granule cells (FD) and CA3 and CA1 pyramidal cells (CA3, CA1). At low magnification there was a general disappearance of the cell layers (E, I, M, Q) compared to control (A), corresponding to the appearance at high magnification of lighter (arrows, K, L, T) or almost absent (arrows, G, H, P) staining of the cytoplasm together with a darker staining of small rounded necrotic nuclei (arrows, F–H, J–L, N–P, R–T).

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B.W. Kristensen et al. / Brain Research 917 (2001) 21 – 44

Fig. 7. (continued)

the induced PI uptake was recorded for all hippocampal subfields together after 2 days of exposure (Fig. 1). When applied to acutely prepared hippocampal slice preparations KA and NMDA have been found to be equally potent [94], whereas KA was more potent than NMDA after intrahippocampal injections in vivo, with AMPA showing an excitotoxic potency in between the two [54]. The relatively low susceptibility to KA might be due to a reduced expression of KA receptors. Speculations on the reason for this may be KA receptor downregulation caused by spontaneous epileptiform activity known to occur in hippocampal slice cultures [58,59,83]. Moreover, repeated long-term application for 3 weeks of 2 mM KA does result in downregulation of GluR6 and KA2 receptor subunit mRNA in the CA3 region of the hippocampal slice cultures and an attenuated susceptibility of CA3 pyramidal cells to 10 mM KA [113].

4.2. Maximal neuronal degeneration Applied in 100 mM concentrations all four glutamate receptor agonists, including ATPA, induced corresponding levels of maximal PI uptake and neuronal degeneration after 2 days of agonist exposure. This allowed the use of EC 50 values of the different compounds after 2 days of exposure to be used as estimates of both their absolute and relative excitotoxic potency. In contrast, it has no meaning to use EC 50 values for comparison of excitotoxic potencies, when for example KA induces total neuronal cell death and AMPA only 50% cell death, as shown in a recent study of murine cortical cultures [52]. Both general cell staining and NeuN- and MAP2-immunostains confirmed that the maximal PI uptake recorded after 2 days of exposure corresponded to total neuronal degeneration in all subfields of the hippocampal slice cultures. Intrahippocam-

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33

Fig. 8. NeuN-immunostained sections of 4-week-old control hippocampal slice culture (A–D) and cultures exposed for 48 h to 100 mM ATPA (E–H), 100 mM AMPA (I–L), 100 mM KA (M–P) or 100 mM NMDA (Q–T). All four glutamate receptor agonists induced distinct degenerative changes in both dentate granule cells (FD) and CA3 and CA1 pyramidal cells (CA3, CA1). At low magnification there was a general decrease in the staining intensity of exposed cultures (E, I, M, Q) compared to control (A) corresponding at high magnification to a lighter staining of the cytoplasm but a darker staining of the nucleus of the neurons (arrows, F–H, J–L, N–P, R–T).

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B.W. Kristensen et al. / Brain Research 917 (2001) 21 – 44

Fig. 8. (continued)

pal injections of AMPA and KA have also been shown to induce widespread and extensive (90–100%) loss of CA1 and CA3 pyramidal cells, as observed in Nissl staining [64]. The same has been found after intrahippocampal injection of NMDA resulting in 70–100% loss of cells in Nissl staining [106]. Also dentate granule cells were severely affected in these studies (50–100% loss of cells in Nissl staining) [64].

4.3. Differential subfield susceptibility to ATPA and AMPA The PI uptake in hippocampal slice cultures exposed to ATPA, AMPA, KA and NMDA revealed mutual differences in susceptibility of the hippocampal subfields (Table 1). When exposed to 30 mM ATPA and 3 mM AMPA, CA1 pyramidal cells were the most susceptible followed by dentate granule cells and CA3 pyramidal cells (CA1.

FD.CA3), suggesting that excitotoxic concentrations of ATPA and AMPA to a large extent act on the same receptors in the three subfields. This was sustained by the observation that GYKI 52466 (and NBQX) had similar neuroprotective effects, when applied together with ATPA or AMPA. Intrahippocampal injections in rats of low doses of AMPA also in particular lesioned CA1 [54]. The observed differences in susceptibility among the hippocampal subfields may be explained by different AMPA receptor densities, which according to binding studies have the following rank order: CA1¯FD.CA3 in the adult hippocampus [36,46,72]. General prediction of differences in susceptibility from binding studies may, however, be difficult since the hippocampal main cell types may express different combinations of AMPA receptor subunits [46,96], which when functionally characterized at the single cell level have been found to interact with AMPA with different potency and efficacy [32,46,96].

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35

Fig. 9. MAP2-immunostained sections of 4-week-old control hippocampal slice culture (A–D) and cultures exposed for 48 h to 100 mM ATPA (E–H), 100 mM AMPA (I–L), 100 mM KA (M–P) or 100 mM NMDA (Q–T). All four glutamate receptor agonists induced distinct degenerative changes in both dentate molecular layer (FD) and stratum radiatum of CA3 and CA1 (CA3, CA1) (E, I, M, Q) compared to control (A). At high magnification a few dendrites in the process of degeneration were found (arrows, K, L, N, P, T). Heavily stained cells bodies were found in most subfields (E, I, M, Q).

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Fig. 9. (continued)

4.4. Differential subfield susceptibility to KA Exposure of hippocampal slice cultures to KA primarily induced degeneration of CA3 pyramidal cells followed by dentate granule cells and CA1 pyramidal cells (CA3. FD.CA1). This rank order confirms earlier reports on KA toxicity in the hippocampus, but the ‘selectivity’ by which CA3 pyramidal cells was lesioned clearly depended on exposure time and KA concentration. Selective CA3 lesions were thus most pronounced at low concentrations (3–8 mM) of KA and long exposures (1–2 days) [6,10,29,87], whereas high 50–100 mM concentrations of KA with short exposure times (30 min to 3 h) resulted in more widespread lesions [9,102]. After exposure to 100 mM KA for 30 min, the most pronounced lesion was thus reported to be in CA1 [102]. Intrahippocampal and intracerebroventricular injections of moderate doses of KA in vivo also preferentially lesion hippocampal CA3

pyramidal cells [4,38,42,47,54,66,68,69], whereas high doses induce degeneration in all subfields [64]. The high susceptibility of CA3 pyramidal cells to KA corresponds to the high density of KA receptors in CA3, with lower densities in fascia dentata and CA1 (CA3.FD.CA1) [2], matching the relative susceptibilities to KA found in the present slice culture study. By contrast, using a 100 mM concentration, KA induced the extensive degeneration in all hippocampal subfields. This may be explained by the action of high concentrations of KA also on AMPA receptors, known from both electrophysiological [79,81] and excitotoxic studies [40,76] of primary cultures of hippocampal and cortical origin.

4.5. Differential subfield susceptibility to NMDA NMDA was found primarily to induce degeneration of CA1 pyramidal cells, followed by dentate granule cells and

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37

Fig. 10. Densities of NeuN-immunostaining (A) measured in the dentate granule cell layer (FDg) and CA3 and CA1 pyramidal cell layers (CA3p, CA1p), and MAP2-immunostaining (B), measured in dentate molecular layer (FDm) and stratum radiatum of CA1 (CA1r) and CA3 (CA3r) of hippocampal control slice cultures and cultures exposed for 48 h to 100 mM ATPA, 100 mM AMPA, 100 mM KA or 100 mM NMDA. Data are shown as means1S.E.M. with n56, *P,0.05, **P,0.01, ***P,0.001, ANOVA with Bonferroni correction for comparison of the different groups.

CA3 pyramidal cells (CA1.FD.CA3). This corresponds to previous qualitative observations where treatment with 10 mM NMDA for 3 h induced PI uptake in CA1 pyramidal cells and dentate granule cells, but only sparse uptake in CA3 [102]. In good accordance with the present results, intrahippocampal injections of 30 nmol NMDA resulted in exactly the same subregional pattern of toxicity: CA1.FD.CA3 [65] matching the different densities of NMDA binding sites found in the 3 subfields in vivo: CA1.FD.CA3 [62,63].

strain differences. Strain differences in seizure responsiveness to KA have thus been reported both for rats [30,31] and mice [25]. In mice, where the KA-induced CA3 pyramidal cell lesion has been demonstrated both in vivo [90] and in vitro [71], certain strains are resistant to KA-induced excitotoxicity [90], just as differences in levels of glutamate receptors [55] and in the distribution of the dentate mossy fiber projection between strains of mice are known [89,91].

4.7. Mechanism of ATPA excitotoxicity 4.6. Species and strain differences in susceptibility to cell death The variations in susceptibility to AMPA, KA and NMDA observed in vivo and in vitro may in part be due to differences in models and protocols, as well as species and

The protective effect of NBQX against 30 mM ATPA was intermediate to the protection against 3 mM AMPA and 8 mM KA. In 1 mM concentration NBQX only partially protected against 30 mM ATPA, while 10 mM NBQX provided complete protection. Compared with

B.W. Kristensen et al. / Brain Research 917 (2001) 21 – 44

38

Table 2 NBQX protected against 30 mM ATPA, 3 mM AMPA, 8 mM KA, but not against 10 mM NMDA, as detected by propidium iodide (PI) uptake in CA1 and CA3 pyramidal cells and fascia dentata granule cells (FD) Agonist

Subfield

NBQX (mM)

30 mM ATPA 30 mM ATPA 30 mM ATPA

CA1 CA3 FD

100.066.9 100.068.4 100.0611.4

3 mM AMPA 3 mM AMPA 3 mM AMPA

CA1 CA3 FD

100.069.9 100.0619.7 100.0621.7

8 mM KA 8 mM KA 8 mM KA

CA1 CA3 FD

100.065.7 100.0611.4 100.0617.9

CA1 CA3 FD

100.068.4 100.069.9 100.0610.6

0

10 mM NMDA 10 mM NMDA 10 mM NMDA

1

10

41.063.1** 47.5610.8*** 86.7610.3

20

2.662.2*** 22.262.4*** 4.367.9***

– – –

9.463.2*** 20.665.6*** 16.2619.3*

3.6610.2*** 21.068.9*** 6.4618.3**

– – –

55.8610.5** 78.3611.6 90.6617.3

55.5610.9** 55.4613.0** 77.5617.5

– – –

91.2622.2 109.7626.9 109.3628.2

26.766.3** 14.764.9*** 21.866.9*** 86.867.4 75.2611.3 85.0614.0

The PI uptake was recorded 48 h after start of exposure to NMDA. The PI uptake without addition of NBQX (0 mM) was set to 100. Data are shown as means6S.E.M. with n511–18, *P,0.05, **P,0.01, ***P,0.001, ANOVA with Bonferroni correction for comparison with control. The four different inhibition data sets for each subfield were significantly different as compared by two-factor ANOVA. Moreover for ATPA, the three different inhibition data set were significantly different for FD in relation to CA1 and CA3.

binding experiments, where NBQX displays a 30-fold displacement of [ 3 H]AMPA binding (IC 50 5150 nM) over [ 3 H]KA binding (IC 50 54.8 mM) [92], this suggests that 30 mM ATPA induced at least 50–60% of its damage in CA1 and CA3, but only limited damage in dentate granule cells, via AMPA receptors. The additional damage, prevented by 10 mM NBQX, might be mediated by KA receptors. Accordingly, also KA receptors could be involved in the excitotoxic effect of ATPA. By contrast, GYKI 52466 was equally protective against 30 mM ATPA and 3 mM AMPA in all hippocampal subfields, pointing to a very limited

involvement of KA receptors in the ATPA-induced excitotoxicity, given that GYKI 52466 displayed an IC 50 value of 18 mM against AMPA receptor-mediated KA currents in cortical neurons, while 200 mM GYKI 52466 inhibited only 30–40% of the KA receptor-mediated KA current in DRG neurons [107]. IC 50 values obtained in other experimental models like primary cultures and single cells may, however, not be directly transferable to hippocampal slice cultures explaining these apparently conflicting results obtained by NBQX and GYKI 52466. The lack of protective effect of 10 mM MK-801 against

Table 3 GYKI 52466 protected against 30 mM ATPA, 3 mM AMPA and 8 mM KA, as detected by propidium iodide (PI) uptake in CA1 and CA3 pyramidal cells and fascia dentata granule cells (FD) Agonist

Subfield

GYKI 52466 (mM) 0

10

30

100

300

91.8613.7 56.2611.1*** 76.1628.6

40.966.7*** 22.564.5*** 42.7613.6**

10.665.3*** 3.362.7*** 6.367.2***

213.366.8*** 212.165.8*** 5.366.3**

30 mM ATPA 30 mM ATPA 30 mM ATPA

CA1 CA3 FD

100.0611.0 100.0610.0 100.0616.4

3 mM AMPA 3 mM AMPA 3 mM AMPA

CA1 CA3 FD

100.0611.4 100.0614.0 100.0614.3

105.3612.8 95.7614.1 130.9619.2

45.4610.6*** 36.667.9*** 71.8614.8**

16.168.7*** 13.963.7*** 44.864.4**

8 mM KA 8 mM KA 8 mM KA

CA1 CA3 FD

100.0616.2 100.069.4 100.0611.7

85.4623.3 67.3613.5 83.0617.3

8.3614.6*** 13.167.9*** 31.1612.0**

19.9625.0* 31.4612.2*** 43.566.0*

6.2613.5*** 5.563.2*** 21.5612.8** 220.668.5* 0.663.5*** 28.9618.8**

The PI uptake was recorded 48 h after start of exposure to AMPA, ATPA or KA. The PI uptake without addition of GYKI 52466 (0 mM) was set to 100. Note that GYKI 52466 was almost equally potent inhibiting ATPA-, AMPA- and KA-induced excitotoxicity. Data are shown as means6S.E.M. with n522–54 for 0–100 mM GYKI 52466 and n56 for 300 mM GYKI 52466. *P,0.05, **P,0.01, ***P,0.001, ANOVA with Bonferroni correction for comparison with control. The three different inhibition data set for each drug and each subfield were not significantly different as compared by two factor ANOVA.

B.W. Kristensen et al. / Brain Research 917 (2001) 21 – 44

39

Table 4 MK-801 protected against 10 mM NMDA, but not against 30 mM ATPA, 3 mM AMPA or 8 mM KA, as detected by propidium iodide (PI) uptake in CA1 and CA3 pyramidal cells and fascia dentata granule cells (FD) Agonist

Subfield

MK-801 (mM)

30 mM ATPA 30 mM ATPA 30 mM ATPA

CA1 CA3 FD

100.069.3 100.0621.9 100.0626.2

– – –

– – –

88.1611.5 108.3638.4 111.0632.2

3 mM AMPA 3 mM AMPA 3 mM AMPA

CA1 CA3 FD

100.067.7 100.0615.0 100.0619.7

– – –

– – –

95.467.1 97.0613.8 121.6621.8

6 mM KA 6 mM KA 6 mM KA

CA1 CA3 FD

100.0614.1 100.0614.5 100.0623.5

– – –

– – –

100.466.8 88.0616.3 85.0623.5

CA1 CA3 FD

100.0614.4 100.0619.4 100.0628.4

0.762.7*** 21.262.9*** 1.269.9***

212.0610.9*** 21.961.9*** 1.2623.5***

0

10 mM NMDA 10 mM NMDA 10 mM NMDA

0.3

3

10

213.466.9*** 22.361.6*** 27.061.7***

The PI uptake was recorded 48 h after start of exposure to ATPA, AMPA, KA or NMDA. The PI uptake without addition of MK-801 (0 mM) was set to 100. Data are shown as means6S.E.M. with n56–22. ***P,0.001, ANOVA with Bonferroni correction for comparison with control. The inhibition data for NMDA were significantly different from the inhibition data for ATPA, AMPA and KA in each subfield as compared by two factor ANOVA. There was no difference between inhibition data for a given agonist in the three different subfields.

30 mM ATPA excluded an NMDA component in the ATPA-induced excitotoxicity in accordance with what was found for AMPA-induced excitotoxicity (see below).

4.8. Mechanism of AMPA excitotoxicity One micromolar NBQX completely protected against 3 mM AMPA-induced damage in both CA1, CA3 and fascia dentata as predicted from its IC 50 value (see above), thereby confirming that AMPA excitotoxicity at this concentration is induced by AMPA receptors. In agreement with this and the IC 50 value of GYKI 52466 (see above), 100 mM concentrations of GYKI 52466 also completely protected against 3 mM AMPA-induced damage in both CA1 and CA3, whereas 300 mM was necessary in the dentate granule cells. MK-801 did not protect against AMPA-induced damage in any of the subfields analyzed. Similar to this the NMDA antagonist (E)-4(3-phosphonoprop-2-enyl)-piperazine-2carboxylic acid, CPP, has been found not to protect against AMPA-induced hippocampal damage in vivo [64]. In primary cultures of cortical neurons, MK-801 has, however, been found to block 88% of the AMPA-induced toxicity, indicating that in this set up most of the AMPAinduced degeneration is mediated indirectly via NMDA receptors. When AMPA receptor desensitization is blocked with cyclothiazide, the major part of the AMPA toxicity is, however, mediated by AMPA receptors and the indirect toxicity via NMDA receptors dramatically reduced [39]. A similar mechanism of AMPA-induced excitotoxicity has been found in primary cultures of hippocampal neurons, when AMPA receptor desensitization is blocked with

cyclothiazide [76]. Without cyclothiazide present, activation of AMPA receptors in primary cultures might accordingly facilitate activation of the NMDA receptors both by lifting the voltage sensitive Mg 21 blockade and through increased activity dependent release of glutamate. The release of glutamate is not important in slice cultures with a high normal or above normal content of astrocytes [20], with a preserved corresponding more efficient glutamate transport ability [88,104]. In agreement with this, millimolar concentrations of glutamate have to be used to induce excitotoxicity in slice cultures [7,104] like in the brain [60], whereas only micromolar concentrations have to be used in primary cultures of cortical neurons [28]. The presently observed potent action of AMPA in hippocampal slice cultures and similar results obtained on striatum and cortex in corticostriatal co-cultures using the same protocol [51], compare well with in vivo studies with almost total neuronal degeneration after injection of AMPA into the hippocampus [54,64] and the striatum [3,56]. By contrast, overnight exposure of primary cultures of hippocampal neurons to 500 mM AMPA was reported only to induce negligible excitotoxicity [57], just as 6 h exposure of cortical neurons to 500 mM AMPA [27,39] and 1 h exposure of striatal neurons to 100–250 mM AMPA [11,12] induced only moderate or no detectable cell death, respectively. The primary cultures used in these studies did, however, all have a low content of astrocytes. One study of cortical neurons showed that a high content of glia increased AMPA excitotoxicty, contrasting other results on the protective effects of glia on NMDA- and glutamateinduced excitotoxicity [23]. Seen in relation to the studies, which have shown that cyclothiazide dramatically en-

40

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hances the lesion produced by AMPA in hippocampal [57] and cortical neurons [39], the findings suggest that the presence of glial cells may influence neuronal AMPA receptor desensitization properties [23]. Another explanation might be that an increased presence of glial cells is responsible for an increased Ca 21 -permeability of AMPA receptors in neurons of the mixed cultures due to a lack of the GluR2 subunit in the AMPA receptor complex [33], making the neurons more susceptible to AMPA. In conclusion, it might thus be the presence of glial cells that influence the neuronal level or composition of AMPA receptors hereby explaining the effects of AMPA in slice cultures and the in vivo situation on one side and in primary cultures on the other side.

4.9. Mechanism of KA excitotoxicity In hippocampal slice cultures exposed to 8 mM KA together with 1 mM NBQX only CA1 was significantly protected (50%). Given that NBQX in this concentration primarily acts on AMPA receptors [92], this suggests an involvement of KA receptors in KA-induced excitotoxicity in both CA1 and CA3 pyramidal cells and dentate granule cells. In accordance with this, the activation of KA receptors, predominantly expressed in CA3, may account for the relatively selective CA3 lesion induced by 8 mM KA and the relatively flat appearance of the concentration– response curves of KA, which are markedly different for ATPA, AMPA and NMDA (Fig. 1). An explanation for the flat concentration–response curves of KA may be that KA at low concentrations first activates KA receptors and then at higher concentrations also AMPA receptors, whereas ATPA, AMPA and NMDA mediate their excitotoxic effects through more homogenous receptor populations. This is in accordance with pharmacological studies, which have shown that KA acts on homomeric GluR6 receptors with an EC 50 of 1 mM [24], on KA receptors in dorsal root ganglion neurons with an EC 50 of 6 mM [108], and on homomeric GluR1 receptors with an EC 50 value of 30–40 mM [19,34] and on AMPA receptors in cortical neurons with an EC 50 value of 160 mM [108]. However, GYKI 52466 was almost equally protective against AMPA- and KA-induced damage in all hippocampal subfields in concentrations of 30–100 mM, where GYKI 52466 is thought to act primarily on AMPA receptors. In excitotoxicity studies in primary cultures of striatal [11], cortical [40] and hippocampal neurons [76], KA elicits the major parts of its toxicity by activation of AMPA receptors, but in these studies 100–500 mM concentrations were used in contrast to a concentration of 8 mM KA used in the present study. KA working also significantly on AMPA receptors in this concentration may, however, be explained by KA producing a largely non-desensitizing response at AMPA receptors [77,80], whereas the response at KA receptors is fast desensitizing [32,48]. Explaining these apparently conflicting results obtained by NBQX and GYKI 52466, it

again seems likely that the pharmacological data obtained in other experimental models like single cells and primary cultures may not be directly transferable to hippocampal slice cultures. There appeared to be no indirect involvement of NMDA receptors in KA-induced excitotoxicity in CA1 and CA3 pyramidal cells or dentate granule cells, as MK-801 was without protective effects in KA exposed slice cultures. The same has been observed in the hippocampus in vivo in one study [26], while another study reported that CPP protected both CA1 pyramidal cells and dentate granule cells against KA, but not CA3 pyramidal cells [64]. In studies on hippocampal neurons, MK-801 has been reported to inhibit 50% of the KA-induced excitotoxicity [76], but in neocortical neurons KA-induced degeneration can only be inhibited to a small extend by MK-801 [40,61] and not by 2-amino-5-phosphonovaleric acid (APV) [14].

4.10. Mechanism of NMDA excitotoxicity MK-801 fully protected neurons in all hippocampal subfields against 10 mM NMDA. By contrast, NBQX was without effect on NMDA-induced excitotoxicity, suggesting that AMPA and KA receptors were not participating in the NMDA-induced cell damage. This corresponds to the findings in vivo that intrahippocampal injection of NBQX together with NMDA was without protective effect [54], and also with other findings in vitro that CNQX and DNQX did not protect against NMDA in primary cultures of cortical neurons [27]. Looking at the time profile for the induced PI uptake, it was clear that NMDA induced degeneration faster than ATPA, AMPA and KA, as demonstrated by the very similar curves for PI uptake after 1 and 2 days. This may be explained by the general high permeability of NMDA receptors to Ca 21 [67,105] compared to AMPA [33,41,46,96] and KA receptors [5,43,70,109]. The relative delay in AMPA / KA receptor-mediated neuronal death, compared to NMDA, may relate to observations in in vivo studies, where delayed treatment with AMPA receptor antagonists, but not NMDA receptor antagonists, can reduce delayed neuronal death in ischemia [21,45,93,111].

5. Concluding remarks By obtaining and comparing concentration–response curves for new (ATPA) and classic (AMPA, KA, NMDA) glutamate receptor agonists and inhibition data for combinations of glutamate receptor agonists and antagonists (NBQX, GYKI 52466, MK-801), we have demonstrated that organotypic hippocampal slice cultures constitute a feasible test system for evaluation of glutamate receptormediated excitotoxic effects and mechanisms. It was revealed that AMPA and ATPA excitotoxicity was mediated primarily via AMPA receptors. However, in low

B.W. Kristensen et al. / Brain Research 917 (2001) 21 – 44

GluR5 selective concentrations, ATPA did not induce excitotoxicity. For KA, the excitotoxicity appeared to be mediated via both KA and AMPA receptors. NMDA receptors were not involved in AMPA-, ATPA- and KAinduced excitotoxicity, nor did NMDA-induced excitotoxicity require activation of AMPA and KA receptors. In summary, slice cultures display more in vivo-like mechanisms of excitotoxicity than reported for primary neuronal cultures, particularly suggesting that slice cultures are preferable for studies where AMPA receptors might be involved.

Acknowledgements The technical help of Inge Holst, Randi Godskesen, Dorte Frederiksen and Margrethe Krog Hansen is gratefully acknowledged. Thanks are also due to Prof. Povl Krogsgaard-Larsen for providing us with ATPA, to Assoc. Prof. Bjarke Ebert for fruitful discussions and help with the manuscript, to Prof. Ferdinando Nicoletti for providing GYKI 52466 and to Prof. Werner Vach for help with statistics. The study was supported by grants from the Danish MRC to Neuroscience Pharmabiotec Research Center, the Aslaug and Carl Friis’s Foundation, the HedeNielsen Foundation, Direktør Jacob Madsens og hustru Olga Madsens Foundation, the Beckett Foundation and the EU-Biotech program (BIO4-CT97-2307).

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