You are here:
Development of a High-Content Image Analysis Method for Quantifying Synaptic Contacts in Rodent Primary Neuronal Cultures
Citation:
JOSHUA, HARRILL A., T. M. FREUDENRICH, B. ROBINETTE, AND W. R. MUNDY. Development of a High-Content Image Analysis Method for Quantifying Synaptic Contacts in Rodent Primary Neuronal Cultures. Presented at TestSmart DNT 2 Meeting, Reston, VA, November 12 - 14, 2008.
Impact/Purpose:
NA
Description:
Development of the nervous system occurs through a series of critical processes, each of which may be sensitive to disruption by environmental contaminants. In vitro culture of neurons can be used to model these processes and evaluate the potential of chemicals to act as developmental neurotoxicants. The present work describes the development of a cell-based high-content analysis (HCA) method for investigating chemical effects on synaptogenesis, a late event in nervous system development. Neocortical neurons (PND0 derived) and cerebellar granule cells (CGCs, PND7 derived) from LE rat pups were seeded at 6.5x104 and 1.5x105 cells/cm2, respectively, on laminin coated 96-well plates. Cells were then fixed at different time points between DIV2 and DIV14 and fluorescently immunostained with Hoescht stain and antibodies against III-tubulin and synapsin I to visualize neuronal nuclei, cell bodies and developing pre-synaptic neurotransmitter vesicles. HCA image acquisition and analysis were performed using a Cellomics® ArrayScan VTiTM automated imaging system and the associated Neural Profiling BioApplication. This analysis program allows for selection of neurons from a mixed cell population, tracing of neuronal cell bodies and neurites and quantification of puncta associated with different compartments of the neuronal cytoplasm. An increase in the number and total area (cm2) of synapsin I puncta associated with neuronal cell bodies and neurites was observed between DIV5 and DIV12. Future research efforts will include examining these measures following exposure to potential positive and negative control compounds and assessment of these endpoints for use as a screening tool for potential developmental neurotoxicants. This abstract does not necessarily reflect USEPA policy.