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Toxicol In Vitro. 2011 Feb;25(1):368-87. doi: 10.1016/j.tiv.2010.10.011. Epub 2010 Oct 20.

Use of high content image analysis to detect chemical-induced changes in synaptogenesis in vitro.

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1
Systems Biology Branch, Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, NC 27711, USA.

Abstract

Synaptogenesis is a critical process in nervous system development whereby neurons establish specialized contact sites which facilitate neurotransmission. Early life exposure to chemicals can result in persistent deficits in nervous system function at later life stages. These effects are often the result of abnormal development of synapses. Given the large number of chemicals in commerce with unknown potential to result in developmental neurotoxicity (DNT), the need exists for assays that can efficiently characterize and quantify chemical effects on brain development including synaptogenesis. The present study describes the application of automated high content image analysis (HCA) technology for examining synapse formation in rodent primary mixed cortical cultures. During the first 15 days in vitro (DIV) cortical neurons developed a network of polarized neurites (i.e., axons and dendrites) and expression of the pre-synaptic protein synapsin increased over time. The localization of punctate synapsin protein in close apposition to dendrites also increased, indicating an increase in synapse formation. Results demonstrated that: (1) punctate synapsin protein with a spatial orientation consistent with synaptic contact sites could be selectively measured, (2) the critical period for synaptogenesis in cortical cultures was consistent with previous reports, (3) chemicals known to inhibit synapse formation decreased automated measurements of synapse number and (4) parallel evaluation of neuron density, dendrite length and synapse number could distinguish frank cytotoxicity from specific effects on synapse formation or neuronal morphology. Collectively, these data demonstrate that automated image analysis can be used to efficiently assess synapse formation in primary cultures and that the resultant data is comparable to results obtained using lower throughput methods.

PMID:
20969947
DOI:
10.1016/j.tiv.2010.10.011
[Indexed for MEDLINE]
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