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Use of high content image analysis to detect chemical-induced changes in synaptogenesis in vitro
Citation:
HARRILL, J., B. ROBINETTE, AND W. R. MUNDY. Use of high content image analysis to detect chemical-induced changes in synaptogenesis in vitro. TOXICOLOGY IN VITRO. Elsevier Science Ltd, New York, NY, 25(1):368-387, (2011).
Impact/Purpose:
In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity (hazard identification). Such screening could facilitate prioritization of resources needed for in vivo toxicity testing towards those chemicals most likely to result in adverse health effects. Cell cultures derived from nervous system tissue can be used in automated, high content screening assays to examine the chemical effects on critical developmental processes. We have previously used high content screening to assess chemical effects on neuronal outgrowth. This study characterizes the use of rat neocortical cells for in vitro assessment of synapse formation, and develops a protocol for automated assessment using high-content image analysis.
Description:
Synaptogenesis is a critical process in nervous system development whereby neurons establish specialized contact sites which facilitate neurotransmission. There is evidence that early life exposure to chemicals can result in persistent deficits in nervous system function, cognition and behavior at later life stages and that these effects are often the result of abnormal development of synapses within various neural networks. 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) on top of a bed of astrocytes. The expression of the pre-synaptic protein synapsin increased over time. The localization of punctate synapsin protein in close apposition to MAP2-positive dendrites also increased, indicating an increase in synapse formation over time. Results of the image analysis demonstrated that: I) 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. This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
