Organotypic Brain Slice Cultures Research Articles

Traumatic injury induces differential expression of cell death genes in organotypic brain slice cultures determined by complementary DNA array hybridization

The expression of a large panel of selected genes hypothesized to play a central role in post-traumatic cell death was shown to be differentially altered in response to a precisely controlled, mechanical injury applied to an organotypic slice culture of the rat brain. Within 48 h of injury, the expression of nerve growth factor messenger RNA was significantly increased whereas the levels of bcl-2, α-subunit of calcium/calmodulin-dependent protein kinase II, cAMP response element binding protein, 65,000 mol. wt isoform of glutamate decarboxylase, 1β isoform of protein kinase C, and ubiquitin messenger RNA were significantly decreased. Because the expression levels of a number of other messenger RNAs such as the neuron-specific amyloid precursor protein, β 2 microglobulin, bax, bcl xl, brain-derived neurotrophic factor, cyclooxygenase-2, interleukin-1β, interleukin-6, tumor necrosis factor-α, receptor tyrosine kinase A, and receptor tyrosine kinase B were unaffected, these selective changes may represent components of an active and directed response of the brain initiated by mechanical trauma. Interpretation of these co-ordinated alterations suggests that mechanical injury to the central nervous system may lead to disruption of calcium homeostasis resulting in altered gene expression, an impairment of intracellular cascades responsible for trophic factor signaling, and initiation of apoptosis via multiple pathways. An understanding of these transcriptional changes may contribute to the development of novel therapeutic strategies to enhance beneficial and blunt detrimental, endogenous, post-injury response mechanisms.

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.

A metallic multisite recording system designed for continuous long-term monitoring of electrophysiological activity in slice cultures

This paper describes a flexible, metallic multielectrode array, made on kapton to fit in a recording chamber for interface-type organotypic cultures. This multisite recording system is designed for continuous multisite monitoring of electrophysiological activity in rat brain organotypic slice cultures. The system is composed of a signal conditioning set-up, which also masters electrical stimulation paradigms and a card containing the microelectrode array. The card comprises a perfusion chamber closed by a rigid and permeable membrane on which the pierced microelectrode array supporting the slice culture is placed. Once closed with a gaseous chamber, the inside of the card remained sterile and free of contamination and could be maintained inside or outside the incubator for electrophysiological analyses. Dimensions of each 28-plated gold microelectrode recording site are 50 μm×100 μm. The design of the chambers and the card makes it possible to modify both the perfusion medium and the gaseous atmosphere in sterile conditions, allowing thus analyses of long-term effects of pharmacological compounds. Using this array one can perform stimulation and recordings of the electrical activity of the slice. Signals obtained with this reusable system exhibit a good signal-to-noise ratio. This device was tested to follow the evolution and modifications of the evoked and/or spontaneous electrical activity of the same groups of neurones during several days .

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 system Topics: Neurotoxicity

Intercellular calcium waves in glia.

Glial cells are capable of communicating increases in [Ca2+]i from a single cell to many surrounding cells. These intercellular Ca2+ waves have been observed in glia in multiple different preparations, including dissociated brain cell cultures, glial cell lines, organotypic brain slice cultures, and intact retinal preparations. They may occur spontaneously, or in response to a variety of stimuli. Ca2+ waves occurring under different conditions in different preparations may have distinctive patterns of initiation and propagation, and distinctive pharmacological characteristics consistent with the involvement of different intracellular and intercellular signaling pathways. This paper presents original data supporting a combination of gap junction and extracellular messenger-mediated signaling in mechanically induced glial Ca2+ waves. Additional new observations provide evidence that a rapidly propagated signal may precede the glial Ca2+ wave and may mediate rapid glial-neuronal communication. This original data is discussed in the context of a review of the literature and current concepts regarding the potential mechanisms, physiological and pathological roles of this dynamic pattern of glial intercellular signaling.

An in vitro blood-brain barrier model: cocultures between endothelial cells and organotypic brain slice cultures.

This communication describes a novel in vitro blood-brain barrier (BBB) model: organotypic slice cultures from the central nervous system were overlaid on endothelial cell monolayers grown on permeable membranes. Morphological, electrophysiological, and microdialysis approaches were carried out to characterize and validate this model. After 10 days in coculture, morphological studies reveal the presence of tight junctions. Electrophysiological recordings of neuronal activity performed on organotypic cultures with or without an endothelial cell monolayer show that amplitude of evoked responses were comparable, indicating good viability of cocultures after 2 weeks. Perfusion of known BBB permeable or nonpermeable molecules was used to test the coculture tightness in conjunction with electrophysiological or microdialysis approaches: application of glutamate (Glu), which doesn't easily cross the BBB, triggers off rhythmic activity only in control cultures, whereas epileptogenic activity was observed in both control cultures and cocultures during perfusions with picrotoxin, a molecule that can diffuse through the BBB. Finally, the microdialysis technique was used to determine the permeability of molecules coming from the perfusion chamber: L-dopa, dopamine, and Glu were employed to assess the selective permeability of the coculture model. Thus, these results indicate that the in vitro model described possesses characteristics similar to those of the BBB in situ and that cocultures of organotypic slices and endothelial cell monolayers have potential as a powerful tool for studying biochemical mechanisms regulating BBB function and drug delivery to the central nervous system.

Trimethyltin (TMT) neurotoxicity in organotypic rat hippocampal slice cultures

The neurotoxic effects of trimethyltin (TMT) on the hippocampus have been extensively studied in vivo. In this study, we examined whether the toxicity of TMT to hippocampal neurons could be reproduced in organotypic brain slice cultures in order to test the potential of this model for neurotoxicological studies, including further studies of neurotoxic mechanisms of TMT. Four-week-old cultures, derived from 7-day-old donor rats and grown in serum-free medium, were exposed to TMT (0.5–100 μM) for 24 h followed by 24 h in normal medium. TMT-induced neurodegeneration was then monitored by (a) propidium iodide (PI) uptake, (b) lactate dehydrogenase (LDH) efflux into the culture medium, (c) cellular cobalt uptake as an index of calcium influx, (d) ordinary Nissl cell staining, and (e) immunohistochemical staining for microtubule-associated protein 2 (MAP-2). Cellular degeneration as assessed by densitometric measurements of PI uptake displayed a dose and time-dependent increase, with the following ranking of vulnerability of the hippocampal subfields: FD>CA4≥CA3c>CA1>CA3ab. This differential neuronal vulnerability observed by PI uptake was confirmed by MAP-2 immunostaining and corresponded to in vivo cell stain observations of rats acutely exposed to TMT. The mean PI uptake of the cultures and the LDH efflux into the medium were highly correlated. The combined results obtained by the different markers indicate that the hippocampal slice culture method is a feasible model for further studies of TMT neurotoxicity.

Organotypic Brain Slice Cultures for Functional Analysis of Alcohol‐Related Disorders: Novel Versus Conventional Preparations

Assessment of long-term alterations in neural function and phenotype has usually involved culture techniques that utilize dissociated preparations. Recently, we have approached such topics in alcohol research by using brain slice cultures, also known as explant or organotypic preparations. In this symposium presentation, two preparations will be discussed, and examples of the particular advantages of these preparations will be presented in relation to alcohol research. First, we use the hippocampal explant preparation for assessment of long-term alterations in N-methyl-D-aspartate receptor (NMDAR) function due to chronic ethanol exposure and subsequent withdrawal. This preparation displays many synaptic, structural, and enzymatic phenotypes indicative of normal neural preparations. Patch clamp recordings reveal NMDAR-mediated excitatory postsynaptic current (EPSC) elicited upon stimulation of Schaffer collateral fibers and recorded from CA1 pyramidal cells. Long-term ethanol exposure followed by subsequent withdrawal resulted in a specific enhancement of NMDAR-mediated synaptic responses which preceded the expression of epileptiform events that occurred after prolonged withdrawal periods. Second, we describe a novel explant preparation, derived from horizontal slices of the entire forebrain and midbrain of the rat. These long-term explants displayed multiple normal phenotypes including Nissl, AChE, TH, and GFAP staining. Electrophysiologically, these explants displayed a functional corticostriatal pathway recorded with field and patch clamp techniques and elicited by synaptic stimulation. Taken together, these explant preparations display utility for long-term study of ethanol effects on neural systems, especially relating to withdrawal hyperexcitability as well as systems involved in drug-seeking behavior.

Organotypic Brain Slice Cultures for Functional Analysis of Alcohol-Related Disorders

Organotypic Brain Slice Cultures for Functional Analysis of Alcohol-Related Disorders

3-Hydroxyglutaric and glutaric acids are neurotoxic through NMDA receptors in vitro.

Glutaryl-CoA dehydrogenase deficiency (GDD; McKusick 231670) causes an accumulation of 3-hydroxyglutaric (30H-GA) and glutaric (GA) acids in body fluids as well as brain tissue (Baric et al 1998; Goodman et al 1977). Typically between the 6th and 18th months of age, intercurrent illness or even a routine immunization can trigger an acute encephalitis-like encephalopathic crisis that leads to an irreversible bilateral striatal destruction and consequently to a severe dystonic-dyskinetic disorder with loss of previously acquired motor skills (Hoffmann et al 1991, 1996). Although the biochemical and clinical features of GDD are well described, the mechanism of age- and regional-specific neuronal damage remains unclear. Substantial evidence points to an excitotoxic sequence in GDD: GA and 30H-GA exhibit structural similarities to the excitotoxic amino acid glutamate, and post-mortem examination of brain tissue of GDD patients revealed postsynaptic vacuolation in striatum and cortex similar to that of glutamate-mediated damage (Goodman et al 1977). A previous study using organotypic rat brain slice cultures showed a neurotoxic effect of 30H-GA, though at high concentrations (> 1.5 mmol/L), but not for GA, suggesting that 30H-GA is the main excitotoxin in GDD (Flott-Rahmel et al 1997). We hypothesized that the age- and regional specific brain damage in GDD could be caused by overstimulation of NMDA receptors and subsequent excitotoxic cell death. We therefore investigated the effects of the main pathological metabolites GA and 30H-GA on neuronal cultures and whether neurotoxic effects could be prevented by NMDA and non-NMDA receptor antagonists.

Development of astrocytes and neurons in cultured brain slices from mice lacking connexin43

Astrocyte and neuronal development was investigated in organotypic brain slice cultures from mouse fetuses with a null mutation in the connexin43 gene. Astrocyte morphology and electrical properties were indistinguishable in null mutant slices and control slices but at 18 days in vitro astrocyte density in the central regions of the null mutant slices was significantly higher than in control slices. Neuronal development assessed morphologically and electrophysiologically appeared normal in the mutant slices. These results suggest that intercellular communication mediated through connexin43 is not essential for the development of astrocytes and neurons but may play a role in regulating astrocytic migration.

Melatonin prevents oxidative stress-induced cell death in hippocampal cells.

The pineal hormone melatonin has been suggested to be a very effective antioxidant in the brain. Oxidative stress may play a role in certain neuropathological conditions and the hippocampus is an early target of neurodegeneration. Using cells of the clonal hippocampal cell line HT22 and organotypic hippocampal rat brain slice cultures, we investigated a possible protective role of melatonin against oxidative stress-induced hippocampal cell death. Glutamate alone induced oxidative apoptotic cell death in HT22 cells as detected with Hoechst staining and DNA fragmentation analysis. Preincubation with 1 mM melatonin protected HT22 cells against glutamate-induced cell death and protected organotypic hippocampal slices against H2O2-induced cell death. These findings suggest that this neurohormone may be useful in the prevention of neurodegenerative diseases.

Workshop on animal models of traumatic brain injury.

Workshop on animal models of traumatic brain injury.

Characterization and application of organotypic brain slice cultures

Characterization and application of organotypic brain slice cultures