There is little knowledge of neurotoxicity (NT) and developmental neurotoxicity (DNT) adverse outcome pathways (AOPs) or the gaps between in vitro Hight Tthroughput Testing (HTT) assays (molecular initiating events (MIEs)/key events (KEs)) and apical outcomes. Developing AOPs for NT /DNT will link KEs (measured in screening assays) to Adverse Outcomes (AO), supporting the use of in vitro data for risk decisions. We are currently focusing on proteomic approaches to examine the role of alterations in synaptogensis and the relationship to AOs. Proteomic signatures relevant to AOP pathways will be examined, including use of genetically-modified cell cultures and in vivo developmental studies. We will manipulate selected mechanisms that are important in brain development (i.e. neurotransmitter systems, hormonal pathways, or neuroanatomical pathways) using multiple techniques (cell culture, zebrafish and rodent models). Optogenetic manipulation of pathways, coupled with multi-electrode recordings, will be used to examine changes in function in "real time". We will then examine the concordence of the results with those of HTT assay's predicted effects. This will allow verification of the biological importance of the AOP and the identification of high priority NT/DNT AOPs allowing focused in vitro and in vivo testing. The work will lead to development of NT/DNT-Relevant AOP(s) based on screening and in vivo outcomes for inclusion in AOP-WIKI.