Opening the black box of traumatic brain injury: a holistic approach combining human 3D neural tissue and an in vitro traumatic brain injury induction device.

Traumatic brain injury (TBI) is caused by a wide range of physical events and can induce an even larger spectrum of short- to long-term pathophysiologies. Neuroscientists have relied on animal models to understand the relationship between mechanical damages and functional alterations of neural cells. These in vivo and animal-based in vitro models represent important approaches to mimic traumas on whole brains or organized brain structures but are not fully representative of pathologies occurring after traumas on human brain parenchyma. To overcome these limitations and to establish a more accurate and comprehensive model of human TBI, we engineered an in vitro platform to induce injuries via the controlled projection of a small drop of liquid onto a 3D neural tissue engineered from human iPS cells. With this platform, biological mechanisms involved in neural cellular injury are recorded through electrophysiology measurements, quantification of biomarkers released, and two imaging methods [confocal laser scanning microscope (CLSM) and optical projection tomography (OPT)]. The results showed drastic changes in tissue electrophysiological activities and significant releases of glial and neuronal biomarkers. Tissue imaging allowed us to reconstruct the injured area spatially in 3D after staining it with specific nuclear dyes and to determine TBI resulting in cell death. In future experiments, we seek to monitor the effects of TBI-induced injuries over a prolonged time and at a higher temporal resolution to better understand the subtleties of the biomarker release kinetics and the cell recovery phases.

SpikeOnChip : A Custom Embedded Platform for Neuronal Activity Recording and Analysis.

In this paper we present SpikeOnChip, a custom embedded platform for neuronal activity recording and online analysis. The SpikeOnChip platform was developed in the context of automated drug testing and toxicology assessments on neural tissue made from human induced pluripotent stem cells. The system was developed with the following goals: to be small, autonomous and low power, to handle micro-electrode arrays with up to 256 electrodes, to reduce the amount of data generated from the recording, to be able to do computation during acquisition, and to be customizable. This led to the choice of a Field Programmable Gate Array System-On-Chip platform. This paper focuses on the embedded system for acquisition and processing with key features being the ability to record electrophysiological signals from multiple electrodes, detect biological activity on all channels online for recording, and do frequency domain spectral energy analysis online on all channels during acquisition. Development methodologies are also presented. The platform is finally illustrated in a concrete experiment with bicuculline being administered to grown human neural tissue through microfluidics, resulting in measurable effects in the spike recordings and activity. The presented platform provides a valuable new experimental instrument that can be further extended thanks to the programmable hardware and software.

Mass Generation, Neuron Labeling, and 3D Imaging of Minibrains.

Minibrain is a 3D brain in vitro spheroid model, composed of a mixed population of neurons and glial cells, generated from human iPSC derived neural stem cells. Despite the advances in human 3D in vitro models such as aggregates, spheroids and organoids, there is a lack of labeling and imaging methodologies to characterize these models. In this study, we present a step-by-step methodology to generate human minibrain nurseries and novel strategies to subsequently label projection neurons, perform immunohistochemistry and 3D imaging of the minibrains at large multiplexable scales. To visualize projection neurons, we adapt viral transduction and to visualize the organization of cell types we implement immunohistochemistry. To facilitate 3D imaging of minibrains, we present here pipelines and accessories for one step mounting and clearing suitable for confocal microscopy. The pipelines are specifically designed in such a way that the assays can be multiplexed with ease for large-scale screenings using minibrains and other organoid models. Using the pipeline, we present (i) dendrite morphometric properties obtained from 3D neuron morphology reconstructions, (ii) diversity in neuron morphology, and (iii) quantified distribution of progenitors and POU3F2 positive neurons in human minibrains.

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

Development and Characterization of PEDOT:PSS/Alginate Soft Microelectrodes for Application in Neuroprosthetics.

Reducing the mechanical mismatch between the stiffness of a neural implant and the softness of the neural tissue is still an open challenge in neuroprosthetics. The emergence of conductive hydrogels in the last few years has considerably widened the spectrum of possibilities to tackle this issue. Nevertheless, despite the advancements in this field, further improvements in the fabrication of conductive hydrogel-based electrodes are still required. In this work, we report the fabrication of a conductive hydrogel-based microelectrode array for neural recording using a hybrid material composed of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate), and alginate. The mechanical properties of the conductive hydrogel have been investigated using imaging techniques, while the electrode arrays have been electrochemically characterized at each fabrication step, and successfully validated both in vitro and in vivo. The presence of the conductive hydrogel, selectively electrodeposited onto the platinum microelectrodes, allowed achieving superior electrochemical characteristics, leading to a lower electrical noise during recordings. These findings represent an advancement in the design of soft conductive electrodes for neuroprosthetic applications.

A VP1 mutation acquired during an enterovirus 71 disseminated infection confers heparan sulfate binding ability and modulates ex vivo tropism.

Enterovirus 71 (EV71) causes hand, foot and mouth disease, a mild and self-limited illness that is sometimes associated with severe neurological complications. EV71 neurotropic determinants remain ill-defined to date. We previously identified a mutation in the VP1 capsid protein (L97R) that was acquired over the course of a disseminated infection in an immunocompromised host. The mutation was absent in the respiratory tract but was present in the gut (as a mixed population) and in blood and cerebrospinal fluid (as a dominant species). In this study, we demonstrated that this mutation does not alter the dependence of EV71 on the human scavenger receptor class B2 (SCARB2), while it enables the virus to bind to the heparan sulfate (HS) attachment receptor and modifies viral tropism in cell lines and in respiratory, intestinal and neural tissues. Variants with VP197L or VP197R were able to replicate to high levels in intestinal and neural tissues and, to a lesser extent, in respiratory tissues, but their preferred entry site (from the luminal or basal tissue side) differed in respiratory and intestinal tissues and correlated with HS expression levels. These data account for the viral populations sequenced from the patient's respiratory and intestinal samples and suggest that improved dissemination, resulting from an acquired ability to bind HS, rather than specific neurotropism determinants, enabled the virus to reach and infect the central nervous system. Finally, we showed that iota-carrageenan, a highly sulfated polysaccharide, efficiently blocks the replication of HS-dependent variants in cells and 2D neural cultures. Overall, the results of this study emphasize the importance of HS binding in EV71 pathogenesis and open new avenues for the development of antiviral molecules that may prevent this virus's dissemination.

Unravelling the effects of multiple experimental factors in metabolomics, analysis of human neural cells with hydrophilic interaction liquid chromatography hyphenated to high resolution mass spectrometry.

This work introduces a strategy for decomposing variable contributions within the data obtained from structured metabolomic studies. This approach was applied in the context of an in vitro human neural model to investigate biochemical changes related to neuroinflammation. Neural cells were exposed to the neuroinflammatory toxicant trimethyltin at different doses and exposure times. In the frame of an untargeted approach, cell contents were analysed using HILIC hyphenated with HRMS. Detected features were annotated at level 1 by comparison against a library of standards, and the 126 identified metabolites were analysed using a recently proposed chemometric tool dedicated to multifactorial Omics datasets, namely, ANOVA multiblock OPLS (AMOPLS). First, the total observed variability was decomposed to highlight the contribution of each effect related to the experimental factors. Both the dose of trimethyltin and the exposure time were found to have a statistically significant impact on the observed metabolic alterations. Cells that were exposed for a longer time exhibited a more mature and differentiated metabolome, whereas the dose of trimethyltin was linked to altered lipid pathways, which are known to participate in neurodegeneration. Then, these specific metabolic patterns were further characterised by analysing the individual variable contributions to each effect. AMOPLS was highlighted as a useful tool for analysing complex metabolomic data. The proposed strategy allowed the separation, quantitation and characterisation of the specific contribution of the different factors and the relative importance of every metabolite to each effect with respect to the total observed variability of the system.

International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes.

A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23-24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.

Engineering of midbrain organoids containing long-lived dopaminergic neurons.

The possibility to generate dopaminergic (DA) neurons from pluripotent stem cells represents an unlimited source of material for tissue engineering and cell therapy for neurodegenerative disease. We set up a protocol based on the generation of size-calibrated neurospheres for a rapid production (3 weeks) of a high amount of DA neurons (>60%) oriented toward a midbrain-like phenotype, characterized by the expression of FOXA2, LMX1A, tyrosine hydroxylase (TH), NURR1, and EN1. By using γ-secretase inhibitors and varying culture time of neurospheres, we controlled maturation and cellular composition of a three-dimensional (3D) engineered nervous tissue (ENT). ENT contained neurons and glial cells expressing various markers of maturity, such as synaptophysin, neuronal nuclei-specific protein (NeuN), and glial fibrillary acidic protein (GFAP), and were electrophysiologically active. We found that 3-week-old neurospheres were optimal to generate 3D tissue containing DA neurons with typical A9 morphology. ENT generated from 4-week-old neurospheres launched glial cell type since astrocytes and myelin could be detected massively at the expense of TH-immunoreactive neurons. All γ-secretase inhibitors were not equivalent; compound E was more efficient than DAPT in generating DA neurons. This DA tissue provides a tool for drug screening, and toxicology. It should also become a useful biomaterial for studies on Parkinson's disease.

Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach.

Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the 'human embryonic stem cell (hESC)-derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (<20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large 'common response' to VPA and MeHg could be distinguished from 'compound-specific' responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles.

OrganDots--an organotypic 3D tissue culture platform for drug development.

INTRODUCTION: There is an urgent need for preclinical testing systems that more accurately reflect responses in human target organs. The use of ex vivo tissues taken out of the human body and kept alive for sufficient time to perform testing has until recently been limited by tissue availability and by the length of time tissues can be kept alive outside the body, however, recent advances in tissue handling and tissue culture techniques have now made it possible to envisage using such tissues for drug discovery on a scale that is of value for the evaluation of compounds prior to testing in humans. AREAS COVERED: The article presents a method for generating 3D microtissues at the air-liquid interface 'OrganDots' which are formed by reaggregating primary tissues or stem cell-based material which may be useful in drug discovery and development. The article compares this method with other methods for obtaining ex vivo tissues and looks at their uses as surrogates to testing compounds in humans. EXPERT OPINION: Reconstituting tissues in vitro has now reached a point where they can be used to profile the activity of compounds prior to in vivo testing. The ability to reconstitute tissues from primary material and the ability to synthesize new tissues in vitro from stem cells may lead to new testing systems that better reflect human pathophysiology and may allow individual differences to be expressed in vitro. These new drug testing systems should lead to more predictable in vitro drug testing systems in the near future.

The long-term survival of in vitro engineered nervous tissue derived from the specific neural differentiation of mouse embryonic stem cells.

Embryonic stem cells (ESCs) offer attractive prospective as potential source of neurons for cell replacement therapy in human neurodegenerative diseases. Besides, ESCs neural differentiation enables in vitro tissue engineering for fundamental research and drug discovery aimed at the nervous system. We have established stable and long-term three-dimensional (3D) culture conditions which can be used to model long latency and complex neurodegenerative diseases. Mouse ESCs-derived neural progenitor cells generated by MS5 stromal cells induction, result in strictly neural 3D cultures of about 120-mum thick, whose cells expressed mature neuronal, astrocytes and myelin markers. Neurons were from the glutamatergic and gabaergic lineages. This nervous tissue was spatially organized in specific layers resembling brain sub-ependymal (SE) nervous tissue, and was maintained in vitro for at least 3.5 months with great stability. Electron microscopy showed the presence of mature synapses and myelinated axons, suggesting functional maturation. Electrophysiological activity revealed biological signals involving action potential propagation along neuronal fibres and synaptic-like release of neurotransmitters. The rapid development and stabilization of this 3D cultures model result in an abundant and long-lasting production that is compatible with multiple and productive investigations for neurodegenerative diseases modeling, drug and toxicology screening, stress and aging research.

Live imaging of remyelination after antibody-mediated demyelination in an ex-vivo model for immune mediated CNS damage.

Mononuclear cell infiltrates, deposits of immunoglobulin and complement as well as demyelination and axonal damage are neuropathological hallmarks of Multiple Sclerosis (MS) lesions. An involvement of antibodies is further suggested by the presence of oligoclonal immunoglobulins in the cerebrospinal fluid of almost all MS patients. However, which mechanisms are most relevant for de- and remyelination and axonal loss in MS lesions is poorly understood. To characterize the regenerative abilities of demyelinated CNS tissue, we utilized murine organotypic cerebellar slice cultures expressing GFP in oligodendrocytes. The addition of a demyelinating monoclonal antibody specific for myelin oligodendrocyte glycoprotein and complement induced complete myelin destruction and oligodendrocyte loss, as demonstrated by confocal live imaging and staining for different myelin proteins. After removal of antibodies and complement we visualized the stages of remyelination, presumably originating from proliferating oligodendrocyte precursor cells and guided by morphologically intact appearing axons. Allowing for the detailed live imaging of de- and remyelination in an ex vivo situation closely resembling the three dimensional cytoarchitecture of the CNS, we provide a useful experimental system for the evaluation of new therapeutic strategies to enhance remyelination and repair in MS.

Development of human nervous tissue upon differentiation of embryonic stem cells in three-dimensional culture.

Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air-liquid interface-based culture of human ESC. This culture system allows three-dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3-month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural-like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air-liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural-like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three-dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues.

Neutralization of the membrane protein Nogo-A enhances growth and reactive sprouting in established organotypic hippocampal slice cultures.

The reduced ability of central axons to regenerate after injury is significantly influenced by the presence of several molecules that inhibit axonal growth. Nogo-A is one of the most studied and most potent of the myelin-associated growth inhibitory molecules. Its neutralization, as well as interference with its signalling, allows for enhanced axonal sprouting and growth following injury. Using differentiated rat organotypic hippocampal slice cultures treated for 5 days with either of two different function-blocking anti-Nogo-A antibodies, we show an increase in CA3 fibre regeneration after lesion. In intact slices, 5 days of anti-Nogo-A antibody treatment led to increased sprouting of intact CA3 fibres that are positive for neurofilament 68. A transcriptomic approach confirmed the occurrence of a growth response on the molecular level upon Nogo-A neutralization in intact cultures. Our results demonstrate that Nogo-A neutralization for 5 days is sufficient for the induction of growth in mature CNS tissue without the prerequisite of an injury. Nogo-A may therefore act as a tonic growth suppressor/stabilizer in the adult intact hippocampus.

Clusterin increases post-ischemic damages in organotypic hippocampal slice cultures.

Clusterin or apolipoprotein J is a heterodimeric glycoprotein which is known to be increased during tissue involution in response to hormonal changes or injury and under circumstances leading to apoptosis. Previous studies in wild-type (WT) and clusterin-null (Clu-/-) mice indicated a protective role of clusterin over-expression in astrocytes lasting up to 90 days post-ischemia. However, in in vitro and in vivo models of neonatal hypoxia-ischemia, clusterin exacerbates necrotic cell death. We developed recombinant forms of clusterin and examined their effect on propidium iodide uptake, neuronal and synaptic markers as well as electrophysiological recordings in hippocampal slice cultures from Clu-/- and WT mice subjected to oxygen-glucose deprivation (OGD). WT mice displayed a marked up-regulation of clusterin associated with electrophysiological deficits and dramatic increase of propidium iodide uptake 5 days post-OGD. Immunocytochemical and western blot analyses revealed a substantial decrease of neuronal nuclei and synaptophysin immunoreactivity that predominated in WT mice. These findings contrasted with the relative post-OGD resistance of Clu-/- mice. The addition of biologically active recombinant forms of human clusterin for 24 h post-OGD led to the abolishment of the ischemic tolerance in Clu-/- slices. This deleterious effect of clusterin was reverted by the concomitant administration of the NMDA receptor antagonist, d-2-amino-5-phosphonopentanoate. The present data indicate that in an in vitro model of ischemia characterized by the predominance of NMDA-mediated cell death, clusterin exerts a negative effect on the structural integrity and functionality of hippocampal neurons.

Electrophysiological recording of re-aggregating brain cell cultures on multi-electrode arrays to detect acute neurotoxic effects.

Neurotoxicity aims to understand how xenobiotics interfere with the function of the nervous system and to unravel their mechanisms of action. Neuronal activity is the primary functional output of the nervous system and deviations from its resting level may indicate toxicity. Consequently, the monitoring of electrophysiological activity in complex cell culture systems appears particularly promising for neurotoxicity assessment. To detect acute neurotoxic effects of chemicals we developed a test system based on the electrophysiological recordings from neural networks in re-aggregating brain cell cultures using multi-electrode arrays. We characterised the electrophysiological properties of the cultures and, using known neurotoxicants, evaluated their usefulness to predict neurotoxic effects. Aggregates displayed evoked field potentials and spontaneous neural activity involving glutamatergic and GABAergic synaptic transmission. Paired pulse inhibition indicated the presence of short-term synaptic plasticity via functional inhibitory networks. Cultures were treated with 0.1-100 microM of trimethyltin chloride (TMT), methyl mercury chloride (MeHgCl), parathion or paraoxon, and with 0.1-100mM of ethanol for up to 100 min. TMT (10 microM), MeHgCl (1 microM) and ethanol (100mM) all decreased the amplitude of evoked field potential. The effect of ethanol was reversible. In contrast paraoxon (10 microM) increased the amplitudes of evoked field potentials while parathion showed no significant effects. The effects of TMT and ethanol on the frequency of spontaneous activity were consistent with those obtained for evoked field potentials. All effects occurred at levels at which cytotoxic injuries were not detectable. Taken together our system expressed electrophysiological properties similar to those of established slice culture preparations. It detected known neurotoxicants at subcytotoxic levels and therefore appears suitable for the assessment of toxic insults specifically interfering with nervous system function, e.g. neuronal activity, synaptic transmission and short-term plasticity. If incorporated into testing strategies, it might represent a valuable tool for the mechanistic assessment of neurotoxic effects.

Interleukin-6 promotes sprouting and functional recovery in lesioned organotypic hippocampal slice cultures.

Interleukin (IL)-6 is a pro-inflammatory cytokine now widely recognized to contribute to the molecular events that follow CNS injury. Little is known, however, about its action on axonal sprouting and regeneration in the brain. We addressed this issue using the model of transection of Schaffer collaterals in mice organotypic hippocampal slice cultures. Transection of slice cultures was associated with a marked release of IL-6 that could be neutralized by an IL-6 blocking antibody. We monitored functional recovery across the lesion by recording synaptic responses using a multi-electrode array. We found that application of IL-6 antibodies to the cultures after lesioning significantly reduced functional recovery across the lesion. Furthermore, the level of expression of the 43-kDa growth-associated protein (GAP-43) was lower in slices treated with the IL-6 neutralizing antibody than in those treated with a control IgG. Conversely, addition of exogenous IL-6 to the culture medium resulted in a dose-dependent enhancement of functional recovery across the lesion and a higher level of expression of GAP-43. Co-culture of CA3 hemi-slices from thy1-YFP mice with CA1 hemi-slices from wild-type animals confirmed that IL-6-treated co-cultures exhibited an increased number of growing fluorescent fibres across the lesion site. Taken together these data indicate that IL-6 plays an important role in CNS repair mechanisms by promoting regrowth and axon regeneration.

Live imaging of neural structure and function by fibred fluorescence microscopy.

Only a few methods permit researchers to study selected regions of the central and peripheral nervous systems with a spatial and time resolution sufficient to image the function of neural structures. Usually, these methods cannot analyse deep-brain regions and a high-resolution method, which could repeatedly probe dynamic processes in any region of the central and peripheral nervous systems, is much needed. Here, we show that fibred fluorescence microscopy-which uses a small-diameter fibre-optic probe to provide real-time images-has the spatial resolution to image various neural structures in the living animal, the consistency needed for a sequential, quantitative evaluation of axonal degeneration/regeneration of a peripheral nerve, and the sensitivity to detect calcium transients on a sub-second timescale. These unique features should prove useful in many physiological studies requiring the in situ functional imaging of tissues in a living animal.

Blockade of NMDA-receptors or calcium-channels attenuates the ischaemia-evoked efflux of glutamate and phosphoethanolamine and depression of neuronal activity in rat organotypic hippocampal slice cultures.

We have investigated the effects of various insults on extracellular glutamate and phosphoethanolamine levels as well as electrical activity alterations in the early period following these insults in organotypic hippocampal slice cultures. Cultures prepared from 7-day-old rats were maintained in vitro for 7-14 days and then metabolic inhibition was induced: cultures were briefly exposed to potassium cyanide to induce chemical anoxia, 2-deoxyglucose with glucose removal to produce hypoglycaemia, or a combination of both to simulate ischaemia. Chemical anoxia induced a small increase in glutamate and a reversible decrease in evoked field potentials and these were greatly potentiated following simulated ischaemia: high, biphasic glutamate efflux and irreversible field potential abolition as well as increase in phosphoethanolamine levels were observed. We have characterised the effects of treatments using NMDA-receptor antagonists and the L-type calcium channel blocker diltiazem. Anoxia-induced glutamate accumulation was prevented by MK-801 and diltiazem D-AP5. Following simulated ischaemia, diltiazem totally prevented glutamate and phosphoethanolamine accumulations, whereas MK-801 did not block the first phase of glutamate accumulation and D-AP5 prevented none. We demonstrated that glutamate and phosphoethanolamine ischaemic-evoked efflux as well as the recovery of electrical activity in organotypic hippocampal slice cultures are sensitive to both NMDA-receptor and calcium-channel blockade. This model thus represents a useful in vitro system for the study of ischaemic neurodegeneration paralleling results reported using in vivo models.