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 and their use in chemical nerve agents. 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 characterize a zebrafish model of OP-induced seizures and excitotoxicity using chlorpyrifos-oxon (CPO)1 – an active metabolite of the common OP pesticide, chlorpyrifos -- and to advance its use for high-throughput compound screening to identify novel MCMs against OP-induced seizures and excitotoxicity. In Aim 1, we will characterize a larval zebrafish model of CPO-induced seizure and optimize parameters for the identification of novel countermeasures. In Aim 1A, we will confirm seizure activity using both electrophysiology (EEG) as well as noninvasive measures based on calcium fluorescence. In addition, we will assess survival and excitotoxicity as evidenced by the burden of apoptotic cells following seizures using TUNEL assay and less time-intensive acridine orange (AO) assay. In Aim 1B, we will identify the ideal assay parameters for detecting novel countermeasures to CPO-induced seizures by identifying the maximum tolerated dose in larval zebrafish of existing countermeasures followed by a CPO dose-response experiment to identify the conditions that result in 25-50% reduction in CPO-induced seizures versus control. This target is chosen to allow detection of the effect of existing countermeasures while providing dynamic range for the detection of superior countermeasures. In Aim 1C, we will quantify sedation as a function of anti-seizure activity in conventional AEDs, by establishing ED50 for several AEDs in response to CPO-induced seizures; these data will be compared to the sedative effect of AEDs in the visual startle response (VSR) and acoustic startle response (ASR) assays. Finally, in Aim 2, we will implement a whole organism compound screen for novel MCMs against OP-induced seizure and excitotoxicity, and prioritize hits based on ranked paired robust strictly standardized mean difference (SSMD*) and least sedating effects. Taken together, this exploratory R21 is responsive to multiple facets of the CounterACT program and provides a framework for whole organism in vivo discovery of novel countermeasures against CPO that may be extended to additional OP agents.