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CONCENTRATION-RESPONSE ASSESSMENT OF CHEMICAL EFFECTS ON SYNAPTOGENESIS USING A HIGH CONTENT IMAGE ANALYSIS-BASED SCREENING ASSAY
Citation:
HARRILL, J., B. ROBINETTE, AND W. R. MUNDY. CONCENTRATION-RESPONSE ASSESSMENT OF CHEMICAL EFFECTS ON SYNAPTOGENESIS USING A HIGH CONTENT IMAGE ANALYSIS-BASED SCREENING ASSAY. Presented at Developmental Neurotoxicity Testing 3 (DNT3) Meeting, Ispra, ITALY, May 10 - 12, 2011.
Impact/Purpose:
These data demonstrate the utility of a high throughput method to rapidly assess chemicals effects on synaptogenesis
Description:
Functional connectivity of the nervous system is dependent upon the development of synapses: i.e. specialized cell-cell contacts which facilitate the unidirectional flow of fast neurotransmission. Prenatal and/or early postnatal exposure to chemicals which disrupt synaptogenesis can result in persistent nervous system deficits in later life. Primary neurons prepared in dissociated culture undergo synaptogenesis in vitro, thereby providing a means to efficiently investigate the effects of chemicals on this critical neuro developmental process. The present work describes development of a high content image analysis-based method for rapid. quantification of synapse formation in primary cultures prepared in a 96well format. Fluorescent labeling of the presynaptic protein synapsin and the dendrite-localized protein MAP2 was used to selectively quantify synaptic puncta in close association with postsynaptic contact sites as an estimator of synapse number. Neuron number and total dendrite length were measured in parallel to assess effects on cell health and morphology. Time course data demonstrated that the critical period of in vitro synaptogenesis identified using automated quantification was similar to that observed. using lower-throughput methods. Chemicals previously shown to inhibit synapse formation in vitro (KCI, Na3V04, mevastatin, bumetanide) decreased synapse number in a concentration-dependent manner. Furthermore, parallel analyses of synapse number, cell number and dendrite length could distinguish specific effects on synapse formation from cytotoxicity. These data demonstrate the utility of a high throughput method to rapidly assess chemicals effects on synaptogenesis. The assay can be incorporated into an in vitro screening battery for developmental neurotoxicity. This abstract does not necessarily reflect USEPA policy.