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ASSESSMENT OF CHEMICAL EFFECTS ON NEURITE OUTGROWTH, NEURONAL POLARIZATION AND SYNAPTOGENESIS IN RAT CORTICAL NEURONS USING HIGH CONTENT IMAGE ANALYSIS
Citation:
ROBINETTE, B., J. HARRILL, AND W. R. MUNDY. ASSESSMENT OF CHEMICAL EFFECTS ON NEURITE OUTGROWTH, NEURONAL POLARIZATION AND SYNAPTOGENESIS IN RAT CORTICAL NEURONS USING HIGH CONTENT IMAGE ANALYSIS. Presented at Society of Toxicology (SOT) Annual Meeting, Washington, DC, March 06 - 10, 2011.
Impact/Purpose:
These data demonstrate that HCA using a single culture system can be used to screen for chemical effects on multiple processes of nervous system development.
Description:
There is a need for efficient, cost-effective methods for screening and prioritization of potential developmental neurotoxicants. One approach uses in vitro cell culture models that can recapitulate the critical processes of nervous system development. In vitro, primary cultures of neocortex undergo a series of morphological changes including neurite outgrowth, polariation of neurites and synaptogenesis similar to that observed in vivo. In the present work, primary cultures were prepared from rat neocortex in a 96-well plate format. A combination of immunocytochemical labeling and highcontent image analysis (HCA) protocols were developed in order to quantify the time course of neurite outgrowth (ßlll-tubulin), neuronal polarization (ß111-tubulin and MAP2 or neurofilament) and synapse formation (MAP2 and synapsin). Neurite outgrowth occurred rapidly: 75 % of neurons developed neurites by 24 h. At this time, ~20% of neurons were axon bearing. An increase in total neurite length continued up 5 days in vitro (DIV), at which time a change in the cytoplasmic distribution if MAP2 was observed.: The ratio of ß111-tubulin and MAP2 labeled neurite lengths (NPR) increased from 1.35 to 2.12 between 24 and 120 h, indicative of neuronal polarization. An increase in synapse number was observed between 6 and 15 DIV. The concentration-response of model chemicals known to inhibit each process was then examined. K252a (0.01 -1 uM) applied at 2 h inhibited total neurite outgrowth (20 %), specifically axon outgrowth (97 %), at 24 h and inhibited neuronal polarization at 120 h (NPR = 1.3). In addition, mevastatin applied at 9 DIV (0.3 -30 uM) decreased synapse number at 15 DIV (~35 %). Effects occurred in the absence of overt cytotoxicity. These data demonstrate that HCA using a single culture system can be used to screen for chemical effects on multiple processes of nervous system development. This abstract does not necessarily reflect USEPA policy.