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Development of in-vitro neural and related immune systems for the study of potentially toxic effects of novel and highly specific compounds during cell differentiation

Objective

To provide technical information to allow the development of in-vitro neural systems capable of detecting the potentially toxic effects of novel and highly specific compounds during cell differentiation. As such sophisticated end points will be used such as receptor-effector coupling, oncogene and transcription factor activation, receptor expression, programmed cell death and cell morphology. Primary cultures, cell lines and brain slices will be used with major emphasis on convulsive and anticonvulsive compounds.
* Sustained induction of the proto-oncogene c-fos has been shown to be a biomarker of excitotoxicity and the basis of an excitotoxicity test.
* Continued work on the role of proto-oncogene expression in cerebellar granule and neocortical neurons stimulated with excitatory aminoacids has suggested that c-fos expression may reflect a neuroprotective response at the level of transcriptional regulation.
* Evaluation of potential end-points for in vitro neurotoxicity screening has shown that, in immature (2 div) and mature (7 div) primary cerebellar cultures, the second messenger molecules cGMP, and the intercellular messenger, nitric oxide, were unsuitable as biomarkers of excitotoxicity.
* Pharmacological analysis of sulphur-amino-acid-stimulated noradrenaline release from hippocampal slice has shown that release is mediated wholly by NMDA receptor activation thus providing an additional potential end-point for monitoring the excitotoxicity of these agents.
* Noradrenaline release in brain slices is a useful model to study pharmacology of agents acting on ionotropic glutamate receptors.
* Cross-talk between metabotropic and ionotropic glutamate receptors in primary cerebellar cultures was demonstrated suggesting such `communication' may be nvolved in adaptive processes such as neuronal development and memory formation, and in neurodegenerative events,
* Demonstration that intracellular calcium store modulators may be of therapeutic potential as neuroprotectants of excitotoxicity.
* Re-appraisal of primary cerebellar cultures as a valid in vitro model to study cGMP formation shows that cGMP is not produced in the neurons but in the astrocytic contaminants of cerebellar cultures. The culture preparation is however shown to be suitable for studying intercellular nitric oxide-cGMP communication.
* End-points on cellular status such as membrane integrity, intracellular Ca++, mitochondrial membrane potential, intracellular free radical formation, have been shown to be valid for assessment of neurotoxicity of organochlorine toxins.
* GABA-induced Cl uptake and flunitrazepam binding in neuronal cultures can differentiate between convulsant and depressant compounds which act on GABA-A receptor Cl channels.
* Although many of the pharmacological and toxic actions of glial and mitochondrial benzodiazepine receptor ligands cannot be correlated to receptor action, receptor binding screening can identify compounds having antiproliferative, prodifferentiative, calcium channel blocking, mitochondrial respiration modulatory and steroidogenic activity.
* A microwell culture system has been devloped to quantify neuronal migration and process development, which can predict teratogenic potential of drugs.
INDUSTRIAL APPLICATIONS:
* Demonstration of a correlation between delayed elevated c-fos expression and excitotoxicity in neuronal cultures. Development of an index of c-fos expression showing high correlation with excitotoxic potential and which can form the basis of a mechanistic screen.
* Neuroteratogenic action of drugs can be predicted using morphometric analysis of cell development in microwell cultures of dissociated tissue from different areas of rat brain.
* Convulsant and depressant drugs acting on GABA-A receptors can be screened using primary cultures of cortical neurons and Cl flux and Flunitrazepam binding assays.
* An in-vitro fluorescent dye bindng assay for neuronal cytotoxicity has been demonstrated.
* Four test proposals are being submitted to ECVAM for consideration for pre-validation or validation.

Funding Scheme

CSC - Cost-sharing contracts
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Coordinator

THE PROVOST, FELLOWS AND SCHOLARS OF THE COLLEGE OF THE HOLY AND UNDIVIDED TRINITY OF QUEEN ELIZABETH NEAR DUBLIN HEREINAFTER TRINITY COLLEGE DUBLIN
Address
College Green
4 Dublin
Ireland

Participants (4)

Bibra International
United Kingdom
Address
Woodmansterne Road
SM5 4DS Carshalton
Consejo Superior de Investigaciones Científicas
Spain
Address
18-26,Jorde Girona Salgado
08034 Barcelona
Danmarks Farmaceutiske Højskole
Denmark
Address
Universitetsparken
2100 København Ø
University of St Andrews
United Kingdom
Address
North Street
KY16 9AL St Andrews