PROJECT SUMMARY Chemical threats are a major public health concern and CounterACT recognizes the need to identify new medical countermeasures (MCMs) that improve the public health response to mass casualty events related to chemical threats. Organophosphate (OP) agents are of particular concern due to their wide availability in common pesticides (e.g. chlorpyrifos, parathion, phorate, aldibcarb) as well as their use in chemical nerve agents (e.g. sarin, soman, tabun, VX). OP-induced seizures are a serious consequence of OP-intoxication and best agents for treating this complication are unclear. Major gaps include limited opportunities for systematic clinical evaluations in humans prior to mass casualty, drawbacks to existing medication, which may not control seizures or excitotoxicity, and worsen sedation; and lack of direction in discovering new MCMs. To address these gaps, we propose to overcome these barriers by harnessing a zebrafish model of OP-induced seizures and excitotoxicity using paraxon-ethyl (PXN) and to advance its use for high-throughput compound screening to identify novel MCMs against OP-induced seizures and excitotoxicity. We hypothesize that zebrafish models of CPO exposure will recapitulate the features of seizures and SE after human exposure to OP agents, which is partly supported by existing literature. Furthermore, we hypothesize that a combination of blind and targeted chemical screening will enable the identification of compounds with novel anti-seizure and/or anti-excitotoxic properties with special efficacy for OP-related seizures. In vivo drug screening most closely approximates the disease pathophysiology and allows optimization for the clinical parameters desired by CounterACT, including post-exposure efficacy, improvement over existing standards-of- care, and minimal sedation for administration without ICU-level monitoring. The following aims will address these hypotheses: Aim 1. Characterize a larval zebrafish model of OP-induced seizure and status epilepticus (SE) and optimize parameters for identification of novel countermeasures. 1A. Confirm seizure activity and excitotoxicity by invasive and non-invasive measures. 1B. Optimize assay parameters to identify novel counter-measures with improved efficacy over existing medical management. 1C. Quantify sedation as a function of anti-seizure activity in conventional AEDs. Aim 2. Whole organism in vivo compound screening for novel countermeasures against OP-induced seizure and excitotoxicity. Taken together, this exploratory R21 is responsive to multiple facets of the CounterACT program including basic mechanistic research to identify molecular mechanisms of acute toxicity of the chemical threat agent chlorpyrifos; creation of an animal model of seizures related to chemical exposure; creation and validation of screening assays for therapy development emphasizing real-world applications to mass casualty events; and identification of candidate therapeutics among FDA-...