Development and characterization of a human embryonic stem cell-derived 3D neural tissue model for neurotoxicity testing.

Alternative models for more rapid compound safety testing are of increasing demand. With emerging techniques using human pluripotent stem cells, the possibility of generating human in vitro models has gained interest, as factors related to species differences could be potentially eliminated. When studying potential neurotoxic effects of a compound it is of crucial importance to have both neurons and glial cells. We have successfully developed a protocol for generating in vitro 3D human neural tissues, using neural progenitor cells derived from human embryonic stem cells. These 3D neural tissues can be maintained for two months and undergo progressive differentiation. We showed a gradual decreased expression of early neural lineage markers, paralleled by an increase in markers specific for mature neurons, astrocytes and oligodendrocytes. At the end of the two-month culture period the neural tissues not only displayed synapses and immature myelin sheaths around axons, but electrophysiological measurements also showed spontaneous activity. Neurotoxicity testing - comparing non-neurotoxic to known neurotoxic model compounds - showed an expected increase in the marker of astroglial reactivity after exposure to known neurotoxicants methylmercury and trimethyltin. Although further characterization and refinement of the model is required, these results indicate its potential usefulness for in vitro neurotoxicity testing.

Multilobular tumor of the zygomatic bone in a dog.

Multilobular tumor of bone (MTB) (also known as Multilobular Osteochondrosarcoma) is an uncommon bone tumor frequently located on the skull of dogs, rarely on the ribs or pelvis. These neoplasms are slow growing, locally invasive, and have the potential to compress and invade the brain. A 10-year-old mixed breed dog was presented with a history of approximately 4 months of progressive growth of a left zygomatic mass. Radiographic investigation revealed a finely granular or stippled non homogeneous radiopaque mass involving the zygomatic arch. After surgery, grossly the neoplasm consisted of multiple, variably sized, grayish-white to yellow nodules separated by collagenous septa of different thickness. Histologically, the tumor was characterized by the presence of multiple lobules containing osteoid and cartilage, separated by a net of fibrous septae. This neoplastic pattern was consistent with a typical multilobular tumor of bone and based on clinical, radiographical, gross and light microscopic findings the definitive diagnosis was made. While reviewing veterinary literature only few cases of MTB were found in dogs.

Embryonic stem cell-based screen for small molecules: cluster analysis reveals four response patterns in developing neural cells.

Neural differentiation of embryonic stem cells (ESC) is considered a promising model to perform in vitro testing for neuroactive and neurotoxic compounds. We studied the potential of a dual reporter murine ESC line to identify bioactive and/or toxic compounds. This line expressed firefly luciferase under the control of the neural cell-specific tubulin alpha promoter (TUBA1A), and renilla luciferase under the control of the ubiquitous translation elongation factor 1-alpha-1 (EEF1A1) promoter. During neural differentiation, TUBA1A activity increased, while EEF1A1 activity decreased. We first validated our test system using the known neurotoxin methyl mercury. This compound altered expression of both reporter genes, with ESC-derived neural precursors being affected at markedly lower concentrations than undifferentiated ESCs. Analysis of a library of 1040 bioactive compounds picked up 127 compounds with altered EEF1A1 and/or TUBA1A promoter activity, which were classified in 4 clusters. Cluster 1 (low EEF1A1 and TUBA1A) was the largest cluster, containing many cytostatic drugs, as well as known neurodevelopmental toxicants, psychotropic drugs and endocrine disruptors. Cluster 2 (high EEF1A1, stable TUBA1A) was limited to three sulfonamides. Cluster 3 (high EEF1A1 and TUBA1A) was small, but markedly enriched in neuroactive and neurotoxic compounds. Cluster 4 (stable EEF1A1, high TUBA1A) was heterogeneous, containing endocrine disruptors, neurotoxic and cytostatic drugs. The dual reporter gene assay described here might be a useful addition to in vitro drug testing panels. Our two-dimensional testing strategy provides information on complex response patterns, which could not be achieved by a single marker approach.

Infection of organotypic slice cultures from rat central nervous tissue with Trypanosoma brucei brucei.

We recently described a new procedure to grow nervous tissue as organotypic culture. The main feature of these slice cultures is to maintain a well preserved, three-dimensional organisation of the central nervous tissue. As these cultures can be kept for several weeks (up to three months), we have used this in vitro approach to study the complex interactions between host tissue and parasites during late stages of cerebral African trypanosomiasis. Light and electron microscopical studies, as well as electrophysiological recordings demonstrate that the structure and function of the nervous tissue is not severely affected even after several weeks of trypanosome infection. The presence of a large number of parasites does not seem to be deleterious to neuronal survival. Secondly, most of the trypanosomes are located around the periphery of the nervous tissue, but many of them also penetrate into the nervous parenchyma. Thirdly, trypanosomes with well-conserved morphology are found within the cytoplasm of glial cells, which in some cases were identified as astrocytes. These "intracellular parasites" seem to actively invade the target cells. Our study demonstrates that the presence of proliferating trypanosomes does not per se interfere with the neural activity of CNS tissues. Secondly, it provides, to the best of our knowledge, the first in vitro demonstration of intracellular forms of African trypanosomes.

Coupling on-line brain microdialysis, precolumn derivatization and capillary electrophoresis for routine minute sampling of O-phosphoethanolamine and excitatory amino acids.

In previous papers, we described the analysis of excitatory amino acids (EAAs) and catecholamines in microdialysis samples using capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD). In the present paper, we report that an automated analysis of such samples can be easily achieved by on-line coupling of the microdialysis probe with a continuous flow derivatization system and a commercially available CE-LIFD apparatus. Because of the short analysis time (less than 2 min) and high separation efficiency (100-200,000 theoretical plates), high temporal resolution of microdialysis (minute range) is preserved as compared to off-line systems, while both EAAs and O-phosphoethanolamine (PEA) can be simultaneously detected. This new method has been applied to the measurement of these compounds in microdialysis samples from hippocampal slice cultures and striatum of anesthetized rats. Extracellular concentrations of EAAs, but not PEA, increased during perfusion of a solution containing high K+ or a glutamate uptake inhibitor. However, after in vitro ischemia on hippocampal slices, both EAAs and PEA concentrations increased, but with different temporal patterns.

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.

Microelectrode arrays for electrophysiological monitoring of hippocampal organotypic slice cultures.

A three-dimensional platinum (Pt) microelectrode array embedded on a micromachined silicon (Si) substrate (porosity of 13%, via hole diameter of 40 microns) has been developed. Electrodes are 35-micron wide and 20-microns high, spaced 200 microns apart and arranged in an elliptic geometry. Integrated within a microperfusion chamber, the devices were used for stimulation and recording experiments of hippocampal slice cultures over a period of several days.

Staurosporine but not chelerythrine inhibits regeneration in hippocampal organotypic cultures.

A mechanical lesion in hippocampal organotypic cultures is followed by a recovery process involving scar formation, sprouting of fibres and formation of new functional synapses. Here we tested the effect of staurosporine and chelerythrine, two protein kinase C (PKC) inhibitors, on this lesion-induced neurite outgrowth of Shaffer collaterals. At a concentration of 1 microM, staurosporine delayed functional recovery assessed by measuring synaptic field potentials across the lesion, without altering synaptic transmission on nonlesioned cultures. Immunostaining carried out by using antibodies directed against neurofilament proteins showed that there was a marked reduction in the number of regenerating fibres crossing the lesion. In contrast to this, chelerythrine (50 microM) did not prevent functional recovery, although it affected synaptic transmission and plasticity at this concentration. We conclude that the inhibition of sprouting produced by staurosporine is independent of its blockade of PKC-mediated phosphorylation mechanisms.

Sprouting and functional recovery in co-cultures between old and young hippocampal organotypic slices.

We developed a model of lesion of Schaffer collaterals in hippocampal organotypic slice cultures to analyse the capacity for sprouting and functional recovery expressed in young (one week old) and old (four week old) slice cultures. Slice cultures were sectioned at different ages of maturation in two separate half-slices and maintained in co-culture. Functional recovery was assessed by measuring synaptic responses elicited across the lesion seven days after the lesion and sprouting was evaluated by biocytin labeling of the regenerating fibers seen under the same conditions. Sprouting and functional recovery were found to be markedly reduced and delayed in old vs young cultures. Preparation of co-cultures between young CA3 and old CA1 half-slices resulted in a significant reduction in the capacity for sprouting and regeneration of the young CA3 neurons. Conversely, co-cultures prepared between old CA3 and young CA1 half-slices showed a markedly enhanced capacity for sprouting and functional recovery of old CA3 neurons. These results indicate that the age-dependent impairment in sprouting and regeneration expressed in cortical regions can be improved by and depends upon the presence of a favourable environment.

Microdialysis monitoring of extracellular glutamate combined with the simultaneous recording of evoked field potentials in hippocampal organotypic slice cultures.

These experiments combined extracellular electrophysiological multirecordings from hippocampal organotypic slice cultures with application of drugs to and sampling of extracellular fluid from a restricted region of the slice using a microdialysis probe. Glutamate (Glu) concentrations were monitored in 0.5 or 2 min microdialysis samples, while evoked field potentials responses (EvFPR) in the CA1 region of the hippocampus (stimulation in the CA3 area) were simultaneously recorded using a multi-electrodes array (Physiocard). Glu was assayed by capillary electrophoresis with laser-induced fluorescence detection combined with a continuous flow derivatization of dialysates. The performance of this combined approach was demonstrated by monitoring extracellular Glu concentrations and EvFPR after K+ induced depolarisation, Glu uptake blockade by trans-pyrrolidine-2,4-dicarboxylic acid (PDC), and electrical stimulation. Such an approach allows a global monitoring of the neuronal functioning with a fine time resolution (up to 30 s) on a simple in vitro brain slice model, to be used as a complement to conventional in vivo microdialysis studies.

A new extracellular multirecording system for electrophysiological studies: application to hippocampal organotypic cultures.

The present paper describes a new multirecording device which performs continuous electrophysiological studies on organotypic cultures. This device is formed by a card (Physiocard) carrying the culture which is inserted into an electronic module. Electrical activities are recorded by an array of 30 biocompatible microelectrodes which are adjusted into close contact with the upper surface of the slice culture. The microelectrode array is integrated into the card enabling electrical stimulation and recording of neurons over periods ranging from several hours to a few days outside a Faraday cage. Neuronal responses are recorded and analyzed by a dedicated electronic and acquisition chain. A perfusion chamber is contained in the card, allowing continuous perfusion in sterile conditions. Electrophysiological extracellular recordings and some drugs' effects obtained with this system in hippocampal slice cultures were identical to conventional electrophysiological set-up results with tetrodotoxin, bicuculline, kainate, dexamethasone and NBQX. The Physiocard system allows new insights for studies on nervous tissue and allows sophisticated approaches to be used quicker and more easily. It could be used for various neurophysiological studies or screening tests such as neural network mapping, nervous recovery, epilepsy, neurotoxicity or neuropharmacology.

Electrophysiological approach of the antiepileptic effect of dexamethasone on hippocampal slice culture using a multirecording system: the Physiocard.

The in vitro antiepileptic activity of the synthetic glucocorticoid dexamethasone (DEX) was tested in rat hippocampal organotypic cultures on the field potential epileptiform activity induced by picrotoxin (PTX). Spontaneous as well as evoked electrophysiological activities have been studied through the extracellular multirecording Physiocard system. PTX typically elicited seizure-like discharges (epileptiform bursts) in the hippocampus neurons. Those epileptiform bursts can be divided in two groups, one rhythmic which lasted 43+/-24s (mean+/-sd) at a frequency of 4.6+/-1.9Hz and the other arhythmic composed of population spikes, which occurred during 14.3+/-6.9min. In the presence of DEX at different concentrations, results obtained were: 1) DEX 1 microM decreased the occurrence of the two different groups of spontaneous epileptiform bursts, most of the time to zero. 2) DEX 50 microM prevented totally the occurrence of epileptiform bursts. 3) DEX 50 microM contrarily to DEX 1 microM avoided the decrease of evoked field potentials' amplitude induced by PTX 3 microM on all simultaneous recorded points. Those results suggest that synthetic glucocorticoid DEX presents an acute antiepileptic effect in a dose dependent manner on the hippocampus tissue.

Enhanced temporal resolution for the microdialysis monitoring of catecholamines and excitatory amino acids using capillary electrophoresis with laser-induced fluorescence detection. Analytical developments and in vitro validations.

This paper reports the development of a method based on capillary electrophoresis with laser-induced fluorescence detection for the simultaneous determination of catecholamines and excitatory amino acids on submicroliter microdialysis samples, with short analysis times (3 min or less), high sensitivity (nanomolar range, i.e., attomoles detected) and high separation efficiency (up to 1.10(6) theoretical plates). A continuous flow derivatization of small volumes of microdialysate (500 nl) using naphthalene-2,3-dicarboxaldehyde as derivatizing reagent is described. Thereafter, two subsequent off-line analyses are performed on each of the 30-s dialysates to determine catecholamines and amino acids. The performances of the present method are demonstrated in vitro by monitoring rapid fluctuations in the concentration of catecholamines and amino acids in the external microdialysis medium.

A new cytochemical method for the ultrastructural localization of calcium in the central nervous system.

We have developed a new cytochemical method for the localization of calcium at the ultrastructural level in the central nervous system (CNS). The method is based on the use of phosphate buffer in the primary fixation followed by a mixture of a complex of chromium(III)-trisoxalate and osmium tetroxide (OsO4) which precipitates calcium and results in the formation of a high electron-dense reaction product. Calcium selectivity was verified by reactions made in test tube, by EGTA treatment of the tissue, by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). The technique was found to be reproducible, yielding similar results in acutely prepared hippocampal slices or organotypic cultures fixed by immersion and in brain areas fixed by perfusion. In hippocampal slices, calcium deposits were found to accumulate in different subcellular compartments such as endoplasmic reticulum, mitochondria and synaptic vesicles. Interestingly, electron-dense reaction products were also visualized in smooth endoplasmic reticulum structures localized in presynaptic terminals or post-synaptic spines as well as in synaptic clefts and active zones. This new method may thus be of interest for studying the metabolism of calcium, specifically with regard to synaptic activity, in the CNS.

A role for polysialylated neural cell adhesion molecule in lesion-induced sprouting in hippocampal organotypic cultures.

By mediating cell-cell interactions, the neural cell adhesion molecule (N-CAM) has been implicated in various events such as axonal pathway formation, neurite outgrowth or synaptic remodelling. One mechanism by which N-CAM could contribute to these events has been proposed to involve modifications of the content of the molecule in polysialic acid. Here we have tested this possibility using an in vitro model of lesion-induced reactive synaptogenesis in hippocampal organotypic cultures. We present evidence that the sprouting reaction triggered by a section of CA3-CA1 connections in these cultures is associated with the expression of the highly sialylated form of N-CAM on regenerating neurites. In addition, we have examined the functional importance of this sialylation mechanism by analysing the effect of treating sectioned cultures with endo-neuraminidase-N which removes the polysialic acid portions of N-CAM. Measurements of the time course of recovery from the lesion, as assessed by the formation of new functional synaptic contacts across the section, showed that removal of the polysialic acid moieties of N-CAM significantly delays the sprouting reaction. The results support the idea that up regulations of highly sialylated forms of N-CAM are of functional importance in neurite sprouting and synapse regeneration in this in vitro model.

Lesion-induced neurite sprouting and synapse formation in hippocampal organotypic cultures.

By sectioning, using a razor blade, one- and three-week-old rat hippocampal organotypic cultures, we have tested the possibility that neurite outgrowth and reactive synaptogenesis would take place even after several weeks in culture in this in vitro model. At the light-microscopic level, recovery from the section and formation of a thin scar were observed within six days following the lesion. Immunostainings using neurofilament antibodies showed the presence of numerous degenerative and regenerative images one day after the cut and many fibres crossing the section six days after the lesion. Electrophysiological recordings of synaptic responses elicited across the section indicated the formation of new functional synaptic contacts and complete recovery of transmission within three to six days. Interestingly, functional recovery in three-week-old cultures was found to be significantly slower than in one-week-old tissue. These findings were confirmed at the electron-microscopic level. Evidence was obtained for an effective cleaning of the lesion site by macrophages and astroglial cells, the existence of many degenerative and regenerative images one day after the cut and the presence of new dendrites, axonal fibres and synapses in the area of the section six days after the lesion. All these changes were slower in three- than in one-week-old cultures. These results indicate that organotypic cultures can be used as an interesting model for studies of reactive synaptogenesis.

Long-term potentiation is associated with an increased activity of Ca2+/calmodulin-dependent protein kinase II.

Among the molecular mechanisms that have been proposed to contribute to long-term potentiation in hippocampus are the activation and autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Here we report that high, but not low frequency stimulation applied to two groups of CA1 afferents resulted in a long lasting increase in the Ca(2+)-independent and total activities of the enzyme as well as an increase in the ratio of Ca(2+)-independent to total activity. The effect was obtained using two different CaM kinase II substrates, it was observed in hippocampal slices and in hippocampal organotypic cultures, and it could be blocked by preincubation of slices with the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonopentanoate. Treatment of slices with calyculin A, a phosphatase inhibitor, modified the activity of the enzyme, but long term potentiation could still be induced and a further increase in Ca(2+)-independent CaM kinase II activity still observed.

Structural modifications associated with synaptic development in area CA1 of rat hippocampal organotypic cultures.

Using morphological techniques, we characterized the developmental reorganization that takes place during the first weeks after explanation in area CA1 of organotypic hippocampal cultures maintained at the interface between medium and a CO2-enriched atmosphere. Pyramidal neurones redistributed from a vertical into an horizontal cell layer in the middle of a three-dimensional culture, with apical dendrites running above the pyramidal layer. Glial cells redistributed into a thin layer at the bottom of the culture, forming an interface between tissue and culture medium. Astrocytes were identified as the most numerous non neuronal cells. No sign of glial proliferation could be observed, except for a transient increase during the first days after explanation. The density of synaptic contacts in the stratum radiatum decreased immediately after explanation and then increased by about 20-fold to reach values in the proximal part of the apical layer after 4 weeks in culture which were only slightly smaller than those measured in 1-month-old rats. The synaptic density in the most distal part of the dendritic layer which receives connections extrinsic to the hippocampus remained significantly lower than in vivo. The ratio of spine to shaft contacts was comparable to that found in vivo. These results indicate that interface type of organotypic cultures can be used as an interesting model for studies of synaptic development in vitro.

Time course of synaptic development in hippocampal organotypic cultures.

Using electrophysiological recordings of field potentials, we investigated the time course of synapse formation and maturation in organotypic cultures prepared from neonate animals of different ages. Following explanation, the size of the maximal synaptic responses elicited in area CA1 by stimulation of a small group of CA3 neurons increased progressively during the first three weeks in culture in a way that corresponded to the changes observed in synaptic contact density. Growth of synaptic responses was found to occur much more rapidly in cultures prepared from 8-day-old as compared with 2-day-old rats. Development of synaptic connections between CA3 and CA1 neurones was also faster than between granule cells and CA3 neurones. Acquisition of mature synaptic properties occurred in vitro as indicated by changes in degree of paired-pulse facilitation and the onset of long-term potentiation (LTP) after a few days in culture. The onset of LTP was much faster in cultures prepared from 8-day-old as compared with 2-day-old neonates and corresponded approximately to the 12-14th postnatal day. It is concluded that development proceeds in the cultures with a time course that resembles the in situ situation.

A simple method for organotypic cultures of nervous tissue.

Hippocampal slices prepared from 2-23-day-old neonates were maintained in culture at the interface between air and a culture medium. They were placed on a sterile, transparent and porous membrane and kept in petri dishes in an incubator. No plasma clot or roller drum were used. This method yields thin slices which remain 1-4 cell layers thick and are characterized by a well preserved organotypic organization. Pyramidal neurons labelled by extra- and intracellular application of horse radish peroxidase resemble by the organization and complexity of their dendritic processes those observed in situ at a comparable developmental stage. Excitatory and inhibitory synaptic potentials can easily be analysed using extra- or intracellular recording techniques. After a few days in culture, long-term potentiation of synaptic responses can reproducibly be induced. Evidence for a sprouting response during the first days in culture or following sections is illustrated. This technique may represent an interesting alternative to roller tube cultures for studies of the developmental changes occurring during the first days or weeks in culture.