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Background: Synaptogenesis is a critical neurodevelopmental process whereby pre- and postsynaptic neurons form apposed sites of contact specialized for chemical neurotransmission. Many neurodevelopmental disorders are thought to reflect altered patterns of synaptic connectivity, including imbalances between excitatory and inhibitory synapses. Developing rapid throughput approaches for assessing synaptogenesis will facilitate toxicologic and drug screening studies of neurodevelopmental disorders. The current study describes the use of high-content imaging to quantify the ontogeny of excitatory and inhibitory synapses using in vitro models of neurodevelopment. These data are compared to biochemical and functional measures of synaptogenesis. Results: The ontogenetic patterns of synapse formation were compared between primary rodent hippocampal and cortical neurons over 28 days in vitro (DIV). As determined by ELISA, the increase in synaptophysin expression levels as cultures matured was similar between hippocampal and cortical cultures. High-content imaging of immunoreactivity of excitatory and inhibitory synaptic biomarkers demonstrated an overall greater number of synapses in hippocampal relative to cortical neurons with marked differences in the pattern of inhibitory synapse development between these two neuronal cell types. Functional assays revealed that both the mean firing rates and mean bursting rates were significantly increased in cortical cultures relative to hippocampal cultures. This difference may reflect decreased inhibitory synaptic tone in cortical versus hippocampal cultures. Conclusions: These data demonstrate differences and similarities in the ontogeny of synaptogenesis between hippocampal and cortical neurons, depending on the biological level examined. Assessment of synaptophysin protein levels by ELISA showed a general increase in synapse formation in both cell types with increasing time in culture, while high-content imaging was able to delineate cell type-dependent differences in formation of excitatory versus inhibitory synapses. The functional significance of differences in the balance of excitatory to inhibitory synapses was confirmed by the assessment of network activity using microelectrode arrays. These results suggest that high-content imaging and microelectrode arrays provide complementary approaches for quantitative assessment of synaptogenesis, which should provide a robust readout of toxicologic and pharmacologic effects on this critical neurodevelopmental event. [ABSTRACT FROM AUTHOR]