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Comparative toxicity induced by domoic acid in immature and mature mixed primarycultures of cerebellar granule cells: involvement of AMPA and NMDA receptors
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
In-Vitro Methods Unit.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Domoic acid is a naturally occurring shellfish toxin that can induce brain damage in mammalians. The toxic effects is thought to be mediated through activation of the AMPA/KA receptor, which induces increased levels of intracellular Ca2+ which in turn cause glutamate release and activates the NMDA receptor. Most studies have been performed in adult animals but neonates have been shown to be more sensitivity to domoic acid per body weight than adults. Prenatal exposure to domoic acid has been associated with damage to neurons in different brain regions, decreased brain GABA levels and increased glutamate levels. In this study we evaluated domoic acid induced toxicity in immature and mature cultures of primary rat cerebellar granule cells (CGCs) by measuring the mRNA levels of selected genes identified as specific glial and neuronal markers. Moreover, we assessed if the induced effects were mediated by activation of the AMPA/KA and/or the NMDA receptor. In addition the influence of the neurotransmitter GABA on domoic acid toxicity was evaluated. The mRNA levels of all the neuronal markers (NF-68, NF-200, NMDA receptor and GABAA receptor) were down-regulated after domoic acid exposure in both immature and mature cultures. However, the mature cultures seemed to be more sensitive to the treatment as the effects were observed at lower concentrations and at an earlier time point than for the immature ones. This could be due to lower expression in young cultures of the receptors that are mediating the toxicity. Indeed, the domoic acid effect could be prevented by the antagonist of the AMPA/KA receptor (NBQX) indicating that this receptor is involved, in contrast to the antagonist for the NMDA receptor (APV) that did not induced any effects. Interestingly, the astrocytic markers (GFAP and S100β) and the neural precursor marker (nestin) were only affected in the mature cultures. These effects could partly be prevented by NBQX, APV and the neurotransmitter GABA, indicating that domoic acid induced toxicity by different mechanisms in astrocytes compared to neurons.

URN: urn:nbn:se:su:diva-30073OAI: diva2:241176
Available from: 2009-10-01 Created: 2009-10-01 Last updated: 2010-01-14Bibliographically approved
In thesis
1. Developmental Neurotoxicity Testing Using In vitro Approaches
Open this publication in new window or tab >>Developmental Neurotoxicity Testing Using In vitro Approaches
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

There is a great concern about children’s health as the developing brain in foetuses and children is much more vulnerable to injury caused by different classes of chemicals than the adult brain. This vulnerability is partly due to the fact that the adult brain is well protected against chemicals by the blood brain barrier (BBB) and children have increased absorption rates and diminished ability to detoxify many exogenous compounds, in comparison to that of adults. Moreover, the development of the central nervous system (CNS) is a very complex process involving several different important events, e.g. proliferation, migration and differentiation of cells. These events are occurring within a strictly controlled time frame and therefore create different windows of vulnerability. Furthermore, the brain consists of numerous different cell types (neuronal, glial and endothelial cells) that have specific functions. The development of each cell type occurs within a specific time window and is therefore susceptible to environmental disturbances at different time periods.

Evidence indicates that exposure to industrial chemicals, pesticides or drugs, contributes to the increasing incidence of neurodevelopment disorders. However, due to lack of studies only a few industrial chemicals have been identified as developmental neurotoxicants so far. The current developmental neurotoxicity (DNT) guidelines (OECD TG 426 and US EPA 712-C-98-239) are based entirely on in vivo studies that are time consuming, complex, costly and not suitable for the testing of a high number of chemicals. Applying alternative approaches such as in silico, in vitro and non-mammalian models as a part of an integrated test strategy, could speed up the process of DNT evaluation and reduce and refine animal usage. Both in vitro and non-mammalian test systems offer the possibility of providing an early screening for a large number of chemicals, and could be particularly useful in characterising the compound-induced mechanism of toxicity of various developmental processes.

This thesis has characterised two primary neuronal cultures (cerebellar granule cells (CGCs) and cortical neuronal cultures) and identified them as relevant models for DNT testing, since the key processes of brain development are present, such as cell proliferation, migration and neuronal/glial differentiation. Furthermore, two emerging technologies (gene expression and electrical activity) have been evaluated and were identified as promising tools for in vitro DNT assessment. In combination with other assays they could be included into a DNT intelligent testing strategy to speed up the process of DNT evaluation mainly by prioritising chemicals with DNT potential for further testing.

Place, publisher, year, edition, pages
Stockholm: The Wenner-Gren Institute, Stockholm University, 2009. 64 p.
National Category
Pharmacology and Toxicology
Research subject
urn:nbn:se:su:diva-30056 (URN)978-91-7155-941-8 (ISBN)
Public defence
2009-10-30, sal E306, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 10:00 (English)
The work of this thesis was performed at ECVAM, European Commission, Italy. At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 3: In progress. Paper 4: In progress. Available from: 2009-10-08 Created: 2009-09-30 Last updated: 2009-10-01Bibliographically approved

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Högberg, Helena T.
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