Activated microglial cells migrate towards sites of excitotoxic neuronal injury inside organotypic hippocampal slice cultures

The aim of this study was to analyse microglial reactions to excitotoxic N ‐methyl‐ d ‐aspartic acid (NMDA)‐induced degeneration of rat dentate and hippocampal neurons in vitro . We used a migration model combining the techniques of microglial single cell culture and organotypic hippocampal slice culture (OHSC). Site‐specific oxidative damage in OHSCs was induced by pretreatment with 50 μ m NMDA. Neuronal injury determined by propidium iodide (PI) uptake included the hippocampal cell layers of the dentate gyrus (DG) and the cornu ammonis (CA). Fluorescence‐prelabelled microglial cells with ameboid morphology were transferred onto the OHSC and migrated predominantly to the prelesioned cell layers of DG and CA when compared with unlesioned areas of the OHSC. In NMDA pretreated slices, microglial cells clustered around degenerating granule cells in the DG and pyramidal cells in the CA. This effect was significantly inhibited in unlesioned slice cultures and in NMDA‐exposed cultures that were pretreated with the NMDA‐antagonist MK‐801. Our observations suggest that microglia – attracted by the presence of stimuli provided by NMDA‐induced neuronal death – migrate specifically towards these lesioned neurons.     

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.     

A method for chronic stimulation of cortical organotypic cultures using implanted electrodes

The neural mechanisms underlying some forms of learning and memory require hours or days to be expressed; however it has proven difficult to study these slowly developing forms of plasticity in reduced preparations due to the short-term nature of acute slice preparations and the fact that most culture preparations lack exposure to structured external input, which plays a critical role in normal cortical development and plasticity. To address this limitation, we developed a method for chronic stimulation of organotypic slice cultures using implanted microelectrodes. This method imparts the ability to apply patterned stimulation to cortical tissue for hours or days, and allows intracellular electrophysiological recordings before and after the stimulation. Importantly, the permanent implantation of the electrodes in the tissue assures that the same neuronal pathways are being excited both during the chronic stimulation while the cultures are in the incubator and while recording in the testing phase. This technique establishes a reduced model for studying experience-dependent plasticity.     

Specific antagonism of excitotoxic action of ‘uncommon’ amino acids assayed in organotypic mouse cortical cultures

Beta-N-Methylamino-L-alanine (BMAA) and beta-N-oxalylamino-L-alanine (BOAA) are chemically related excitant amino acids present in the seeds of Cycas circinalis and Lathyrus sativus, respectively. Consumption of these seeds has been linked to Guam amyotrophic lateral sclerosis (BMAA) and lathyrism (BOAA) (a form of primary lateral sclerosis). We report that the acute neuronotoxic actions of these amino acids are blocked selectively by specific glutamate receptor antagonists. Administration of BOAA and BMAA to neonatal mouse cortex explants (EC100 = 28 microM and 1.6 mM, respectively) rapidly induces postsynaptic vacuolation (PSV) and neuronal degeneration characterized by dark/shrunken (D/S) cells. BOAA-mediated neuronotoxic effects are attenuated in a concentration-dependent manner by cis-2,3-piperidine dicarboxylic acid (PDA), an antagonist of quisqualate (QA)-preferring and kainate (KA)-preferring glutamate receptors. PDA maximally protected against BOAA-induced PSV by 84% at 1 mM and D/S cells by 80% at 0.5 mM. BMAA-induced cellular changes were antagonized selectively in a concentration-dependent manner by 2-amino-7-phosphono-heptanoic acid (AP7), an N-methyl-D-aspartate (NMDA) glutamate-receptor antagonist. AP7 maximally protected against BMAA-induced PSV and D/S by 88% at 1.0 and 0.5 mM, respectively. These protective actions were selective and specific since AP7 failed to attenuate BOAA-induced alterations, and PDA was ineffective in ameliorating BMAA-induced changes. Other glutamate receptor antagonists (glutamic diethyl ester and streptomycin) failed to protect the explants from the destructive action of either toxin. Taken collectively, our data indicate that the acute neuronotoxic actions of BOAA and BMAA (or a metabolite) operate through different glutamate receptor species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic multichannel recordings from organotypic hippocampal slice cultures: protection from excitotoxic effects of NMDA by non-competitive NMDA antagonists

In vitro neuronal damage has traditionally been evaluated by biochemical or anatomical but not by electrophysiological techniques. In the present study, we combined two newly developed technologies, an 8×8 multi-electrode array (MED-64) and cultured hippocampal slices, to demonstrate the potential use of electrophysiological measures as index of neuronal damage. We first demonstrated the stability of electrophysiological recordings over prolonged periods of time (up to 14 days) in field CA1 of cultured hippocampal slices following electrical stimulation of the Schaffer collateral pathway. We then assessed the neurotoxic properties of NMDA and AMPA and determined that the time-course, potency, and efficacy of these two neurotoxins were similar to those assessed by other experimental approaches. We also compared the efficacy and potency of two non-competitive NMDA receptor antagonists to protect against NMDA-mediated neurotoxicity. Again, the results matched well with the results obtained from traditional techniques. Thus, this new technology might provide a new and powerful method to study the chronic effects of drugs or other experimental manipulations in an in vitro preparation.     

Human fetal myelinated organotypic cultures

We have previously reported the establishment of organotypic cultures derived from human fetal brain tissue. Although these cultures permit the testing of multiple hypotheses about normal human neurodevelopment and neuropathologic conditions, they have the limitation of not being myelinated and therefore preclude the study of questions related to myelinogenesis and diseases of myelin. In the current communication, we describe recent developments that allow us to overcome this limitation and permit the establishment of a myelinated organotypic culture model. Sections of dorsal column dissected from the lumbar spinal cord of human fetuses ranging in age 21-23 weeks of gestation were placed in culture. The explants were maintained for up to 12 weeks during which time they were characterized and shown to express a number of CNS cell-type-specific markers including glial fibrillary acidic protein (astrocytes), nerve growth factor receptor and neurofilament protein (neurons), CD68 (microglia), and myelin basic protein, HNK-1 and galactocerebroside (oligodendrocytes). In addition, lectin histochemistry using Ricinus communis agglutinin-1 detected microglia and endothelial cells. Upon explantation, abundant myelin was seen by electron microscopy in the cultures. Although during the culture period there was degradation of myelin, there was also evidence of maintenance of intact myelin sheaths around small caliber axons and de novo myelin synthesis. This model system may permit the further use of human organotypic cultures to investigate issues related to neurodevelopment and to pathologic conditions including those relevant to dysmyelination and demyelination.     

Dopaminergic stimulation in unilateral neglect

OBJECTIVE—To explorethe hypothesis that dopaminergic circuits play a part in the premotorcomponents of the unilateral neglect syndrome, the effects of acutedopaminergic stimulation in patients with neglect were studied.
METHODS—Two tasks wereevaluated before and after subcutaneous administration of apomorphineand placebo: a circle crossing test and a test of target exploration (amodified version of the bell test), performed both in perceptual(counting) and in perceptual-motor (pointing) conditions.
SUBJECTS—Four patientswith left neglect.
RESULTS—Afterdopaminergic stimulation, a significant improvement was found comparedwith placebo administration and baseline evaluation, in the performanceof the two tests. Three of the patients had a more marked improvementin the perceptual-motor condition (pointing) of the task than theperceptual condition (counting).
CONCLUSIONS—Thefindings suggest that dopaminergic neuronal networks may mediate, indifferent ways, both perceptive and premotor components of theunilateral neglect syndrome.

     

Calbindin-D 28K in hippocampal organotypic cultures

Slices of hippocampus from 6-day-old rats were cultured for 2-4 weeks using the roller-tube technique. The organization of these explants was studied by immunocytochemical labeling of calbindin-D 28K (CaBP 28K). The development of the CaBP 28K staining was very close to that of the rat hippocampus in vivo with only 3 subpopulations of labeled cells: granule cells and their mossy fibers, pyramidal cells in the subiculum-CA1 zone and interneurons scattered in strata oriens and radiatum.     

Interleukin-1beta exacerbates and interleukin-1 receptor antagonist attenuates neuronal injury and microglial activation after excitotoxic damage in organotypic hippocampal slice cultures

The effects of interleukin (IL)-1beta and IL-1 receptor antagonist (IL-1ra) on neurons and microglial cells were investigated in organotypic hippocampal slice cultures (OHSCs). OHSCs obtained from rats were excitotoxically lesioned after 6 days in vitro by application of N-methyl-D-aspartate (NMDA) and treated with IL-1beta (6 ng/mL) or IL-1ra (40, 100 or 500 ng/mL) for up to 10 days. OHSCs were then analysed by bright field microscopy after hematoxylin staining and confocal laser scanning microscopy after labeling of damaged neurons with propidium iodide (PI) and fluorescent staining of microglial cells. The specificity of PI labeling of damaged neurons was validated by triple staining with neuronal and glial markers and it was observed that PI accumulated in damaged neurons only but not in microglial cells or astrocytes. Treatment of unlesioned OHSCs with IL-1beta did not induce neuronal damage but caused an increase in the number of microglial cells. NMDA lesioning alone resulted in a massive increase in the number of microglial cells and degenerating neurons. Treatment of NMDA-lesioned OHSCs with IL-1beta exacerbated neuronal cell death and further enhanced microglial cell numbers. Treatment of NMDA-lesioned cultures with IL-1ra significantly attenuated NMDA-induced neuronal damage and reduced the number of microglial cells, whereas application of IL-1ra in unlesioned OHSCs did not induce significant changes in either cell population. Our findings indicate that: (i) IL-1beta directly affects the central nervous system and acts independently of infiltrating hematogenous cells; (ii) IL-1beta induces microglial activation but is not neurotoxic per se; (iii) IL-1beta enhances excitotoxic neuronal damage and microglial activation and (iv) IL-1ra, even when applied for only 4 h, reduces neuronal cell death and the number of microglial cells after excitotoxic damage.     

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 μM) for 24 h or the excitotoxic glutamate agonist kainic acid (KA) (5–25 μM) 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 μM) 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.Themes: Disorders of the nervous systemTopics: Neurotoxicity     

Function of microglia in organotypic slice cultures.

We have examined the functional characteristics of microglia in an environment where the cytoarchitecture of the brain is preserved. Using organotypic slice culture under serum-free conditions, microglia initially demonstrated a rounded morphology but after 10 days in vitro (DIV), microglia in the slice were highly branched. Stimulation of the microglia at 4 DIV with phorbol ester significantly increased the number of cells stained with nitroblue tetrazolium, an indicator of superoxide anion production, compared to non-stimulated conditions. At 10 DIV, superoxide anion production was significantly less than that seen at 4 DIV and no increase in production was seen with phorbol ester stimulation. Phagocytosis of fluorescent latex beads and chemotaxis of microglia in response to zymosan activated serum was also reduced at 10 DIV compared to 4 DIV. These experiments indicate that microglia at 4 DIV in tissue slice culture have functional characteristics that resemble microglia in primary culture. However, prolonged culture of the slices results in a return of the microglia to a ramified and functionally down-regulated state, reminiscent of an "in vivo"-like environment. The organotypic slice culture, thus, provides a useful model system to I examine the interactions of microglia with neurons and other glia in the normal and injured brain.     

Bismuth tracing in organotypic cultures of rat hippocampus

Bismuth is known to have neurotoxic side effects in humans and animals. In the 1970s France experienced about a thousand cases of patients suffering from bismuth-induced encephalopathy. Studies suggest that bismuth may provoke a selective degeneration of CA1 pyramidal cells in the organotypic cultures of rat hippocampus. A currently established technique for the histochemical visualization of bismuth was applied on hippocampal tissue cultures allowing the tracing of bismuth in concentrations hitherto not possible. The accumulation and subcellular localization of bismuth is demonstrated in the tissue cultures of rat hippocampus. CA1 pyramidal cells in the rat hippocampus exhibit the highest uptake of bismuth. High bismuth citrate concentrations (10 microM) are able to totally destroy the cytoarchitecture of the hippocampus. At ultrastructural levels bismuth was found to be located exclusively in lysosome-like organelles.     

Colchicine induces apoptosis in organotypic hippocampal slice cultures

The microtubule-disrupting agent colchicine is known to be particular toxic for certain types of neurons, including the granule cells of the dentate gyrus. In this study we investigated whether colchicine could induce such neuron-specific degeneration in developing (1 week in vitro) and mature (3 weeks in vitro) organotypic hippocampal slice cultures and whether the induced cell death was apoptotic and/or necrotic. When applied to 1-week-old cultures for 48 h, colchicine induced primarily apoptotic, but also a minor degree of necrotic cell death in the dentate granule cells, as investigated by cellular uptake of the fluorescent dye propidium iodide (PI), immunostaining for active caspase 3 and c-Jun/AP-1 (N) and fragmentation of nuclei as seen in Hoechst 33342 staining. All four markers appeared after 12 h of colchicine exposure. Two of them, active caspase 3 and c-Jun/AP-1 (N) displayed a similar time course and reached a maximum after 24 h of exposure, 24 h ahead of both PI uptake and Hoechst 33342 staining, which together displayed similar time profiles and a close correlation. In 3-week-old cultures, colchicine did not induce apoptotic or necrotic cell death. Attempts to interfere with the colchicine-induced apoptosis in 1-week-old cultures showed that colchicine-induced PI uptake and formation of apoptotic nuclei were temporarily prevented by coapplication of the protein synthesis inhibitor cycloheximide. Application of the pancaspase inhibitor z-VAD-fmk almost completely abolished the formation of active caspase 3 protein and apoptotic nuclei induced by colchicine, but the formation of necrotic nuclei increased correspondingly and the PI uptake was unaffected. We conclude that colchicine induces caspase 3-dependent apoptotic cell death of dentate granule cells in hippocampal brain slice cultures, but the apoptotic cell death is highly dependent on the developmental stage of the cultures.     

Calcium-binding protein immunocytochemistry in organotypic cultures of cerebellum

Vitamin D-dependent calcium-binding protein is known to be present in specific classes of neurons including Purkinje cells of the rat and chick. Expiant cultures of newborn mouse cerebellum consisting of cerebellar cortex and deep nuclear region were fixed at maturity (20 days in vitro). Paraffin sections were reacted with the antiserum to purified chick duodenal calcium-binding protein. The Purkinje cell of these cultures reacted in its entirety—neuronal soma, dendrite, axon and terminals (in the deep nuclear region). The results verified many of the findings of our previous morphological studies. Qualitatively similar results were obtained with cultures maintained either in medium containing serum and embryo-extract or in a defined medium. There is not yet sufficient data to indicate whether this protein in the neurons in culture is dependent upon an exogenous source of vitamin D. This immocytochemical marker should prove useful to identify a specific neuron-type in culture.     

Human fetal central nervous system organotypic cultures

Many aspects of human neurodevelopment remain enigmatic. A main reason for this is, although there have been a significant number of morphologic and biochemical studies of neural tissues derived from human embryos and fetuses, this can only provide a static picture of the events at a given gestational age. Also, in vitro studies that focus on cells derived from these tissues have a limitation in that different cell types in dissociated tissue culture cannot interact in a 'normal' physiologic manner thereby, perhaps, altering their housekeeping and luxury functions. The present study focused on the development of a human explant organotypic culture model that may overcome the static limitation of the first example and permit a dynamic analysis of different cell types as they interact which may satisfy the second restriction. Because there is an array of developmental markers that define different cell phenotypes, this explant model may also provide a means of analyzing, for the first time, processes undefined in the human CNS. Human fetal CNS tissue obtained from second trimester abortuses was established in culture. The tissues were maintained for up to 12 weeks during which time they developed and differentiated. Sample cultures were harvested periodically and analyzed by light- and electron microscopy in combination with immunocytochemistry. Differentiation of neurons, astrocytes, oligodendrocytes and endothelial cells was documented using morphologic and biochemical criteria. As such, this model system may allow for the analysis of processes that occur during normal human fetal neurodevelopment and in pathologic conditions.     

A chemically-defined medium for organotypic slice cultures

Organotypic slice cultures provide an excellent system for the analysis of study of the molecular mechanisms of this development necessitates the use of a chemically defined culture medium. We report here the development of a medium, EOL1 defined medium, designed specifically for this purpose. Cultures of both cerebral cortex and basal forebrain demonstrate that this defined medium allows a high degree of cytoarchitectural maintenance while promoting neural metabolism and process outgrowth.     

Dye coupling in Purkinje cells of organotypic slice cultures

Cerebellar slice cultures of newborn rats showed poorly developed dendritic arborization of Purkinje cells, whereas cultures of 10-day-old rats revealed prominent dendritic branching. Gap junctional intercellular communication between Purkinje cells, investigated as dye transfer of microinjected neurobiotin, occurred through dendro-dendritic contacts, with decreased dye spreading in old cell cultures. These results indicate a possible correlation of gap junctional intercellular communication and the development of Purkinje cells.     

Rhythm generation in organotypic medullary cultures of newborn rats

Organotypic transverse medullary slices (obex level) from six-day-old rats, cultured for two to four weeks in chemically defined medium contained rhythmically discharging neurones which were activated by CO₂ and H⁺. The mechanisms underlying this rhythmicity and the spread of excitation and synaptic transmission within this organotypic tissue were examined by modifying the composition of the external solution. Our findings showed that (1) Exposure to tetrodotoxin (0.2 μM) or to high magnesium (6 mM) and low calcium (0.2 mM) concentrations abolished periodic activity. (2) Neither the blockade of GABAergic potentials with bicuculline methiodide (200 μM) and/or hydroxysaclofen (200 μM) nor the blockade of glycinergic potentials with strychnine hydrochloride (100 μM) abolished rhythmicity. (3) While atropine sulphate (5 μM) was ineffective in modulating periodic discharges nicotine (100 μM) — like CO₂ — shortened the intervals between the periodic events; hexamethonium (50–100 μM) reduced both periodic and aperiodic activity. (4) Exposure to the NMDA antagonist 2-aminophosphonovaleric acid (50 μM) suppressed periodic events only transiently. In the presence of 2-aminophosphonovaleric acid rhythmicity recovered. However, the AMPA-antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10–50 μM), abolished periodic activity reversibly within less than 5 min. When 6-cyano-7-nitroquinoxaline-2,3-dione and nicotine were administered simultaneously periodic events persisted for up to 10 min. These findings indicate that synaptic excitatory drive is a prerequisite for the generation of rhythmic discharges of medullary neurones in this preparation. This drive may activate voltage-dependent channels or it may facilitate endogenous cellular mechanisms which initiate oscillations of intracellular calcium concentration. To test the latter possibility (5) calcium antagonists were added to the bath saline. The organic calcium antagonists verapamil and flunarizine (50–100 μM each) and the inorganic calcium antagonists cobalt (2 mM) and magnesium (6 mM) suppressed periodic activity and abolished or weakened the chemosensitivity towards CO₂/acidosis. (6) Dantrolene (10 μM), an inhibitor of intracellular calcium release decreased the periodicity, while thapsigargin (2 μM) which blocks endoplasmic Ca²⁺-ATPase, transiently accelerated the occurrence of periodic events. (7) Oscillations of intracellular free calcium concentrations in Fura-2 AM-loaded cells were weakened or abolished by cobalt (2 mM). The results of (5)–(7) indicate that transmembrane calcium fluxes as well as intracellular Ca²⁺-release and -clearance mechanisms are a prerequisite for intracellular free calcium oscillations which may be important in the generation of rhythmic discharges in medullary neurones.     

Cyclic GMP modulators and excitotoxic injury in cerebral cortical cultures

N-Methyl-D-aspartate (NMDA) receptor activation generates nitric oxide (NO) and cyclic GMP (cGMP) and produces 'excitotoxic' neuronal injury. To examine the possible role of cGMP in excitotoxicity, we evaluated the effects of agents that stimulate or inhibit cGMP activity on the release of lactate dehydrogenase from neuron-enriched cortical cultures. cGMP analogs exhibited no toxicity, and inhibitors of guanylate cyclase or of cGMP-dependent enzymes failed to protect cultures from the toxic effects of NMDA or the NO donor sodium nitroprusside. These findings argue against a role for cGMP in the pathogenesis of excitotoxic neuronal injury.