DiscussionLow level exposure to some toxicants can affect neuronal structure leading to developmental and cognitive impairment. Synaptic transmission is the fundamental mechanism of communication between neurons. Here, we used human induced- pluripotent stem cell (hiPSC) neurons grown in 384-well dishes for up to 15 days to examine the effects of toxicant exposure on synapses in a high content screen (HCS). These cells demonstrate action potentials as early as 9 days and sensitivity to chemical inhibitors as early as 12 days. To detect synapses, we used antibodies to the pre- and post-synaptic proteins, Synapsin-1 and Post-Synaptic Density protein 95 (PSD95), respectively.
To analyze synapse response to toxicant exposure, we analyzed fixed images using algorithms to mask neurite regions marked by expression of β-III tubulin and measured colocalized signals for the pre- and post- synaptic markers only in these functionally-relevant regions. Our HCS approach has enabled us to screen a library of EPA ToxCAST compounds, FDA-approved drugs (the NCC2 library), as well as other agents, such as glutamate and glycine, and Brefeldin A, known to modulate neuronal function. Additional information includes the amount of neurite outgrowth, and alterations in nuclear texture; these may be related to the mechanism of action for molecules that show effects in the assay. Using this approach, we have developed a robust platform for large-scale screening of chemicals that affect synapse formation, the basic unit of neuronal function in humans.