Targeted next-generation sequencing of adult gliomas for retrospective prognostic evaluation and up-front diagnostics.

AIMS: We aimed to reclassify a population-based cohort of 529 adult glioma patients to evaluate the prognostic impact of the 2016 World Health Organization (WHO) central nervous system tumour classification. Moreover, we evaluated the feasibility of gene panel next-generation sequencing (NGS) in daily diagnostics of 225 prospective glioma patients. METHODS: The retrospective cohort was reclassified according to WHO 2016 criteria by immunohistochemistry for IDH-R132H, fluorescence in situ hybridization for 1p/19q-codeletion and gene panel NGS. All tumours of the prospective cohort were subjected to NGS analysis up-front. RESULTS: The entire population-based cohort was successfully reclassified according to WHO 2016 criteria. NGS results were obtained for 98% of the prospective patients. Survival analyses in the population-based cohort confirmed three major prognostic subgroups, that is, isocitrate dehydrogenase (IDH)-mutant and 1p/19q-codeleted oligodendrogliomas, IDH-mutant astrocytomas and IDH-wildtype glioblastomas. The distinction between WHO grade II and III was prognostic in patients with IDH-mutant astrocytoma. The survival of patients with IDH-wildtype diffuse astrocytomas carrying TERT promoter mutation and/or EGFR amplification overlapped with the poor survival of IDH-wildtype glioblastoma patients. CONCLUSIONS: Gene panel NGS proved feasible in daily diagnostics. In addition, our study confirms the prognostic role of glioma classification according to WHO 2016 in a large population-based cohort. Molecular features of glioblastoma in IDH-wildtype diffuse glioma were linked to poor survival corresponding to IDH-wildtype glioblastoma patients. The distinction between WHO grade II and III retained prognostic significance in patients with IDH-mutant diffuse astrocytic gliomas.

Brain tumour diagnostics using a DNA methylation-based classifier as a diagnostic support tool.

AIMS: Methylation profiling (MP) is increasingly incorporated in the diagnostic process of central nervous system (CNS) tumours at our centres in The Netherlands and Scandinavia. We aimed to identify the benefits and challenges of MP as a support tool for CNS tumour diagnostics. METHODS: About 502 CNS tumour samples were analysed using (850 k) MP. Profiles were matched with the DKFZ/Heidelberg CNS Tumour Classifier. For each case, the final pathological diagnosis was compared to the diagnosis before MP. RESULTS: In 54.4% (273/502) of all analysed cases, the suggested methylation class (calibrated score ≥0.9) corresponded with the initial pathological diagnosis. The diagnosis of 24.5% of these cases (67/273) was more refined after incorporation of the MP result. In 9.8% of cases (49/502), the MP result led to a new diagnosis, resulting in an altered WHO grade in 71.4% of these cases (35/49). In 1% of cases (5/502), the suggested class based on MP was initially disregarded/interpreted as misleading, but in retrospect, the MP result predicted the right diagnosis for three of these cases. In six cases, the suggested class was interpreted as 'discrepant but noncontributory'. The remaining 33.7% of cases (169/502) had a calibrated score <0.9, including 7.8% (39/502) for which no class indication was given at all (calibrated score <0.3). CONCLUSIONS: MP is a powerful tool to confirm and fine-tune the pathological diagnosis of CNS tumours, and to avoid misdiagnoses. However, it is crucial to interpret the results in the context of clinical, radiological, histopathological and other molecular information.

Molecular pathology of tumors of the central nervous system.

Since the update of the 4th edition of the WHO Classification of Central Nervous System (CNS) Tumors published in 2016, particular molecular characteristics are part of the definition of a subset of these neoplasms. This combined 'histo-molecular' approach allows for a much more precise diagnosis of especially diffuse gliomas and embryonal CNS tumors. This review provides an update of the most important diagnostic and prognostic markers for state-of-the-art diagnosis of primary CNS tumors. Defining molecular markers for diffuse gliomas are IDH1/IDH2 mutations, 1p/19q codeletion and mutations in histone H3 genes. Medulloblastomas, the most frequent embryonal CNS tumors, are divided into four molecularly defined groups according to the WHO 2016 Classification: wingless/integrated (WNT) signaling pathway activated, sonic hedgehog (SHH) signaling pathway activated and tumor protein p53 gene (TP53)-mutant, SHH-activated and TP53-wildtype, and non-WNT/non-SHH-activated. Molecular characteristics are also important for the diagnosis of several other CNS tumors, such as RELA fusion-positive subtype of ependymoma, atypical teratoid rhabdoid tumor (AT/RT), embryonal tumor with multilayered rosettes, and solitary fibrous tumor/hemangiopericytoma. Immunohistochemistry is a helpful alternative for further molecular characterization of several of these tumors. Additionally, genome-wide methylation profiling is a very promising new tool in CNS tumor diagnostics. Much progress has thus been made by translating the most relevant molecular knowledge into a more precise clinical diagnosis of CNS tumors. Hopefully, this will enable more specific and more effective therapeutic approaches for the patients suffering from these tumors.

Prognostic value of O-6-methylguanine-DNA methyltransferase (MGMT) protein expression in glioblastoma excluding nontumour cells from the analysis.

AIMS: It is important to predict response to treatment with temozolomide (TMZ) in glioblastoma (GBM) patients. Both MGMT protein expression and MGMT promoter methylation status have been reported to predict the response to TMZ. We investigated the prognostic value of quantified MGMT protein levels in tumour cells and the prognostic importance of combining information of MGMT protein level and MGMT promoter methylation status. METHODS: MGMT protein expression was quantified in tumour cells in 171 GBMs from the population-based Region of Southern Denmark (RSD)-cohort using a double immunofluorescence approach. Pyrosequencing was performed in 157 patients. For validation we used GBM-patients from a Nordic Study (NS) investigating the effect of radiotherapy and different TMZ schedules. RESULTS: When divided at the median, patients with low expression of MGMT protein (AF-low) had the best prognosis (HR = 1.5, P = 0.01). Similar results were observed in the subgroup of patients receiving the Stupp regimen (HR = 2.0, P = 0.001). In the NS-cohort a trend towards superior survival (HR = 1.6, P = 0.08) was seen in patients with AF-low. Including MGMT promoter methylation status, we found for both cohorts that patients with methylated MGMT promoter and AF-low had the best outcome; median OS 23.1 and 20.0 months, respectively. CONCLUSION: Our data indicate that MGMT protein expression in tumour cells has an independent prognostic significance. Exclusion of nontumour cells contributed to a more exact analysis of tumour-specific MGMT protein expression. This should be incorporated in future studies evaluating MGMT status before potential integration into clinical practice.

Tumour-associated microglia/macrophages predict poor prognosis in high-grade gliomas and correlate with an aggressive tumour subtype.

AIMS: Glioblastomas are highly aggressive and treatment resistant. Increasing evidence suggests that tumour-associated macrophages/microglia (TAMs) facilitate tumour progression by acquiring a M2-like phenotype. Our objective was to investigate the prognostic value of TAMs in gliomas using automated quantitative double immunofluorescence. METHODS: Samples from 240 patients with primary glioma were stained with antibodies against ionized calcium-binding adaptor molecule-1 (IBA-1) and cluster of differentiation 204 (CD204) to detect TAMs and M2-like TAMs. The expression levels were quantified by software-based classifiers. The associations between TAMs, gemistocytic cells and glioblastoma subtype were examined with immuno- and haematoxylin-eosin stainings. Three tissue arrays containing glioblastoma specimens were included to study IBA-1/CD204 levels in central tumour and tumour periphery and to characterize CD204+ cells. RESULTS: Our data revealed that the amount of especially CD204+ TAMs increases with malignancy grade. In grade III-IV, high CD204 expression was associated with shorter survival, while high IBA-1 intensity correlated with a longer survival. In grade IV, CD204 showed independent prognostic value when adjusting for clinical data and the methylation status of O6-methylguanine-DNA methyltransferase. Our findings were confirmed in two bioinformatics databases. TAMs were more abundant in central tumour tissue, mesenchymal glioblastomas and gliomas with many gemistocytic cells. CD204+ TAMs co-expressed proteins related to tumour aggressiveness including matrix metallopeptidase-14 and hypoxia-inducible factor-1α. CONCLUSIONS: This is the first study to use automated quantitative immunofluorescence to determine the prognostic impact of TAMs. Our results suggest that M2-like TAMs hold an unfavourable prognostic value in high-grade gliomas and may contribute to a pro-tumourigenic microenvironment.

Acute hypoxia induces upregulation of microRNA-210 expression in glioblastoma spheroids.

AIM: Tumor hypoxia and presence of tumor stem cells are related to therapeutic resistance and tumorigenicity in glioblastomas. The aim of the present study was therefore to identify microRNAs deregulated in acute hypoxia and to identify possible associated changes in stem cell markers. MATERIALS & METHODS: Glioblastoma spheroid cultures were grown in either 2 or 21% oxygen. Subsequently, miRNA profiling was performed and expression of ten stem cell markers was examined. RESULTS: MiRNA-210 was significantly upregulated in hypoxia in patient-derived spheroids. The stem cell markers displayed a complex regulatory pattern. CONCLUSION: MiRNA-210 appears to be upregulated in hypoxia in immature glioblastoma cells. This miRNA may represent a therapeutic target although it is not clear from the results whether this miRNA may be related to specific cancer stem cell functions.

Improved histopathological evaluation of gliomas using tissue fragments obtained by ultrasonic aspiration.

Ultrasonic aspirators are commonly used to resect brain tumors because they allow safe, rapid and accurate removal of dissected tissue. However, the tissue fragments removed by ultrasonic aspirators are used surprisingly little in daily diagnostics and in clinical research. A comparison between diagnoses made on the tissue fragments removed by the Sonocut ultrasonic aspirator and the tissue in conventional tumor biopsies was made. The correspondence between the amount of Sonocut tissue analyzed and the probability of making the most malignant diagnosis was analyzed statistically in order to limit the amount of tissue fragments, which need to be analyzed, and in order to save tissue for other purposes like molecular genetics and in vitro drug sensitivity assays. Thirty cases were included in the present study and consisted of astrocytomas, glioblastomas, oligodendrogliomas and ependymomas. The results showed that in 8 out of 30 cases only the Sonocut tissue fragments contained the tumor components that provided the most malignant diagnosis. In further 20 cases, the Sonocut tissue fragments and conventional tumor biopsies gave the same diagnoses. In the remaining 2 cases, the most malignant foci were included in the biopsy removed for peroperative frozen section investigation. When the slides with Sonocut tissue fragments were analyzed, the probability of making the most malignant diagnosis increased from 81.3% - 99.1%, when slides from 1 - 5 paraffin blocks were analyzed, respectively. When subgroups of small, medium and big tumors were analyzed, it was found that only 2 paraffin blocks from small tumors need to be prepared to reach 98.3% probability of making the most malignant diagnosis, whereas 5 paraffin blocks from big tumors need to be prepared to reach a 96.8% probability. In conclusion, the study shows that a limited amount of Sonocut ultrasonic tissue fragments improve the diagnostic evaluation of gliomas. These tissue fragments therefore must not be discarded. Only few paraffin blocks need to be prepared to reach close to 100% probability of making the most malignant diagnosis, reducing the amount of slides, which have to be analyzed and saving Sonocut tissue fragments for future use in molecular genetics and drug sensitivity assays.

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

The excitotoxic profiles of (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA), (RS)-2-amino-3-hydroxy-5-methyl-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)=KA (13 mM)>ATPA (33 mM). Exposed to 30 microM ATPA, 3 microM AMPA and 10 microM NMDA, CA1 was the most susceptible subfield followed by fascia dentata and CA3. Using 8 microM KA, CA3 was the most susceptible subfield, followed by fascia dentata and CA1. In 100 microM 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 microM). In low GluR5 selective concentrations (0.3-3 microM), 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.

The metabotropic glutamate receptor agonist 1S,3R-ACPD stimulates and modulates NMDA receptor mediated excitotoxicity in organotypic hippocampal slice cultures.

The potential toxic effects of the metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) and its interactions with the N-methyl-D-aspartate (NMDA) receptor were studied in hippocampal brain slice cultures, using densitometric measurements of the cellular uptake of propidium iodide (PI) to quantify neuronal degeneration. Cultures exposed to ACPD, showed a concentration (2-5 mM) and time (1-4 days) dependent increase in PI uptake in CA1, CA3 and dentate subfields after 24 h and 48 h of exposure, with CA1 pyramidal cells being most sensitive. The neurodegeneration induced by 2 mM ACPD was completely abolished by addition of 10 microM of the NMDA receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), while 20 microM of the 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainic acid receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) had no effect. Co-exposing cultures to a subtoxic dose of 300 microM ACPD together with 10 microM NMDA, which at this dose is known to induce a fairly selective degeneration of CA1 pyramidal cells, significantly increased the PI uptake in both CA1 and CA3, compared to cultures exposed to 10 microM NMDA only. Adding the 300 microM ACPD as pretreatment for 30 min followed by a 30 min wash in normal medium before the ACPD/NMDA co-exposure, eliminated the potentiation of NMDA toxicity. The potentiation was also blocked by addition of 10 or 100 microM 2-methyl-6-(phenylethynyl)pyridine (MPEP) (mGluR5 antagonist) during the co-exposure, while a corresponding addition of 10 or 100 microM 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (mGluR1 antagonist) had no effect. We conclude that, stimulation of metabotropic glutamate receptors with ACPD at concentrations of 2 mM or higher induces a distinct subfield-related and time and concentration dependent pattern of hippocampal degeneration, and that ACPD at subtoxic concentrations modulates NMDA-induced excitotoxicity through the mGluR5 receptor in a time dependent way.

Biocompatibility of silicon-based arrays of electrodes coupled to organotypic hippocampal brain slice cultures.

In this study we examined the passive biocompatibility of a three-dimensional microelectrode array (MEA), designed to be coupled to organotypic brain slice cultures for multisite recording of electrophysiological signals. Hippocampal (and corticostriatal) brain slices from 1-week-old (and newborn) rats were grown for 4-8 weeks on the perforated silicon chips with silicon nitride surfaces and 40 microm sized holes and compared with corresponding tissue slices grown on conventional semiporous membranes. In terms of preservation of the basic cellular and connective organization, as visualized by Nissl staining, Timm sulphide silver-staining, microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) immunostaining, the slice cultures grown on chips did not differ from conventionally grown slice cultures. Neither were there any signs of astrogliosis or neurodegeneration around the upper recording part of the 47-microm-high platinum-tip electrodes. Slice cultures grown on a separate set of chips with platinum instead of silicon nitride surfaces also displayed normal MAP2 and GFAP immunostaining. The width of the GFAP-rich zone (glia limitans) at the bottom surface of the slice cultures was the same ( approximately 20 microm) in cultures grown on chips with silicon nitride and platinum surfaces and on conventional insert membranes. The slice cultures grown on chips maintained a normal, subfield differentiated susceptibility to the glutamate receptor agonist N-methyl-D-aspartate (NMDA) and the neurotoxin trimethyltin (TMT), as demonstrated by the cellular uptake of propidium iodide (PI), which was used as a reproducible and quantifiable marker for neuronal degeneration. We conclude that organotypic brain slice cultures can grow on silicon-based three-dimensional microelectrode arrays and develop normally with display of normal subfield differentiated susceptibilities to known excito- and neurotoxins. From this it is anticipated that the set-up, designed for recording of electrophysiological parameters, can be used for long-term studies of defined neuronal networks and provide valuable information on both normal, neurotoxicological and neuropathological conditions.

Excitatory amino acid neurotoxicity and modulation of glutamate receptor expression in organotypic brain slice cultures.

Using organotypic slice cultures of hippocampus and cortex-striatum from newborn to 7 day old rats, we are currently studying the excitotoxic effects of kainic acid (KA), AMPA and NMDA and the neuroprotective effects of glutamate receptor blockers, like NBQX. For detection and quantitation of the induced neurodegeneration, we have developed standardized protocols, including--a) densitometric measurements of the cellular uptake of propidium iodide (PI), --b) histological staining by Flouro-Jade, --c) lactate dehydrogenase (LDH) release to the culture medium, --d) immunostaining for microtubulin-associated protein 2, and --e) general and specific neuronal and glial cell stains. The results show good correlation between the different markers, and are in accordance with results obtained in vivo. Examples presented in this review will focus on the use of PI uptake to monitor the excitotoxic effects of --a) KA and AMPA (and NMDA) in hippocampal slice cultures, and --b) KA and AMPA in corticostriatal slice cocultures, with demonstration of differentiated neuroprotective effects of NBQX in relation to cortex and striatum and KA and AMPA. A second set of studies include modulation of hippocampal KA-induced excitotoxicity and KA-glutamate receptor subunit mRNA expression after long-term exposure to low, non-toxic doses of KA and NBQX. We conclude that organotypic brain slice cultures, combined with standardized procedures for quantitation of cell damage and receptor subunit changes is of great potential use for studies of excitotoxic, glutamate receptor-induced neuronal cell death, receptor modulation and related neuroprotection.

GDNF and neublastin protect against NMDA-induced excitotoxicity in hippocampal slice cultures.

The potential neuroprotective effects of glial cell line-derived neurotrophic factor (GDNF) and neublastin (NBN) against NMDA-induced excitotoxicity were examined in hippocampal brain slice cultures. Recombinant human GDNF (25-100 ng/ ml) or NBN, in medium conditioned by growth of transfected, NBN-producing HiB5 cells, were added to slice cultures I h before exposure to 10 microM NMDA for 48h. Neuronal cell death was monitored, before and during the NMDA exposure, by densitometric measurements of propidium iodide (PI) uptake and loss of Nissl staining. Both the addition of rhGDNF and NBN-containing medium significantly reduced the NMDA-induced PI uptake in the CA1 (p < 0.01), suggesting neuroprotective effects of these factors, beyond their well-known trophic effects on dopaminergic neurons.

Excitotoxic effects of non-NMDA receptor agonists in organotypic corticostriatal slice cultures.

The excitotoxic effects of the glutamate receptor agonists kainic acid (KA) and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and the corresponding neuroprotective effects of the AMPA/KA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) were examined in corticostriatal slice cultures. The purpose was to examine the feasibility of these cultures for excitotoxic studies, and to demonstrate possible differential excitotoxic effects of KA and AMPA on striatal and cortical neurons. Slices of dorsolateral striatum with overlying neocortex were obtained from neonatal rats and grown on semiporous membranes in serum-free medium for 3-4 weeks before exposure to KA or AMPA for 48 h. The uptake by injured cells of the fluorescent dye propidium iodide (PI) added to the culture medium was used as a quantifiable measure for neuronal degeneration and compared with efflux of the cytosolic enzyme lactate dehydrogenase (LDH) into the culture medium and loss of glutamic acid decarboxylase (GAD) activity in the tissue. Histological sections were also stained by the fluorescent dye Fluoro-Jade (FJ), for degenerating neurons and by immunocytochemical staining for gamma-aminobutyric acid (GABA). Digitized images showed a dose (0-24 microM KA, 0-6 microM AMPA) and time (0-48 h) dependent increase in PI uptake in both striatum and cortex. In other cultures exposed to KA (24 microM) or AMPA (6 microM) together with NBQX (0.1-9 microM), NBQX was found to exert a differential neuroprotective effect on striatum and cortex at low doses. NBQX was thus more protective against KA in the cortex than in the striatum, while the opposite was seen in relation to AMPA. Regarding neurodegenerative markers, PI uptake was significantly correlated with (1) LDH release into the culture medium, (2) optical density of Fluoro-Jade staining, (3) loss of GAD-activity in tissue homogenates, and (4) loss of GABA-immunostained neurons. We conclude that both differences between compounds (AMPA vs. KA) and brain areas (striatum vs. cortex) can be demonstrated in corticostriatal slice cultures, which in conjunction with an established set of markers for neuronal cell damage appears to be a feasible model for studies of the neurotoxic and neuroprotective effects of glutamate receptor agonists and antagonists.

Coupling of organotypic brain slice cultures to silicon-based arrays of electrodes.

Fetal or early postnatal brain tissue can be cultured in viable and healthy condition for several weeks with development and preservation of the basic cellular and connective organization as so-called organotypic brain slice cultures. Here we demonstrate and describe how it is possible to establish such hippocampal rat brain slice cultures on biocompatible silicon-based chips with arrays of electrodes with a histological organization comparable to that of conventional brain slice cultures grown by the roller drum technique and on semiporous membranes. Intracellular and extracellular recordings from neurons in the slice cultures show that the electroresponsive properties of the neurons and synaptic circuitry are in accordance with those described for cells in acutely prepared slices of the adult rat hippocampus. Based on the recordings and the possibilities of stimulating the cultured cells through the electrode arrays it is anticipated that the setup eventually will allow long-term studies of defined neuronal networks and provide valuable information on both normal and neurotoxicological and neuropathological conditions.

Markers for neuronal degeneration in organotypic slice cultures.

This protocol describes ways of monitoring spontaneous or induced neuronal degeneration in organotypic brain slice cultures. Hippocampal cultures (4-week-old) are grown in normal serum-free control medium, or exposed to the neurotoxin trimethyltin (TMT) (0.5-100 microM) for 24 h or the excitotoxic glutamate agonist kainic acid (KA) (5-25 microM) for 48 h followed by 24 h or 48 h, respectively, in normal medium. Corticostriatal slice cultures (also 4-week-old) are exposed to KA (6-24 microM) for 48 h and normal medium for control. The resulting neurodegeneration is estimated by (a) propidium iodide (PI) uptake, (b) lactate dehydrogenase (LDH) efflux to the culture medium, (c) ordinary Nissl cell staining, (d) staining by the neurodegenerative marker Fluoro-Jade (FJ), (e) neuronal microtubule degeneration by immunohistochemical staining for microtubule-associated protein 2 (MAP2), and (f) Timm sulphide silver staining for heavy metal alterations. Both hippocampal and corticostriatal slice cultures show a dose- and time-dependent increase in PI uptake and LDH efflux after exposure to TMT and KA. The mean PI uptake and the LDH efflux into the medium correlate well for both types of cultures. Both TMT and KA exposed hippocampal cultures display in vivo patterns of differential neuronal vulnerability as evidenced by PI uptake, FJ staining and MAP2 immunostaining. Corticostriatal slice cultures exposed to a high dose of KA display extensive striatal and cortical degeneration in FJ staining as suggested by a high PI uptake. A change in Timm sulphide silver staining in deep central parts of some control cultures, corresponds to areas with loss of cells in cell staining, loss of MAP2 staining, PI uptake, and FJ staining. We conclude that organotypic brain slice cultures, in combination with appropriate markers in standardized protocols, represent feasible means for studies of excitotoxic and neurotoxic compounds.