Our objective in this project is to determine the destructive seizure-inducing effects of the systemically administered organophosphate diisopropyl fluorophosphate (DFP) on rat brain, and to determine the roles of the necroptotic and exitotoxic pathways of programmed necrosis in DFP- induced neuronal necrosis. For Specific Aim 1 we will determine the time course and degree of neuronal necrosis (using the hematoxylin and eosin [H & E] stain) and nuclear translocation of receptor-interacting protein (kinase)-1 (RIP1), receptor-interacting protein (kinase)-3 (RIP3) and mixed lineage kinase domain-like protein (MLKL), using immunofluorescence microscopy, 6, 24 and 72 h after DFP administration in dorsal hippocampal CA1-CA3 and hilus. For Specific Aim 2 we will determine if co-administration of diazepam (the current standard of care) with either the RIP1 inhibitor 7-Cl-O-necrostatin-1 (7-Cl-O-nec-1) or the NMDA-receptor antagonist MK-801 (dizocilpine), given 60 min after DFP injection, are neuroprotective and if 7-Cl-O-nec-1 reduces nuclear translocation of RIP1, RIP3 and MLKL in the hippocampus 24 h (or 6 or 72 h, depending on the results of Specific Aim 1) after DFP injection. For Specific Aim 3 we will determine if co-administration of diazepam and both 7-Cl-O-nec-1 and MK-801 60 min after DFP injection provides greater hippocampal neuroprotection than either given by itself. For Specific Aim 4 we will determine if 7-Cl-O-Nec-1 and/or MK-801 (depending on the results of Specific Aims 2 and 3) provides greater neuroprotection and better spatial learning and memory in DFP-treated rats compared to those given vehicle two one months after DFP-induced status epilepticus (SE). The methods that we will use are (1) in situ transcardiac perfusion-fixation of the brain with 4% phosphate-buffered paraformaldehyde for light and immunofluorescence microscopy; (2) the stereological optical fractionator for unbiased estimates of hippocampal CA1-3 and hilar neurons after 60-µm-thick coronal sections are stained with H & E for light microscopy (Specific Aims 1-4) or RIP1, RIP3 and MLKL antibodies for immunofluorescence microscopy (Specific Aim 2); (3) subcellular fractionation and western blots as a separate approach to confirm the degree of nuclear translocation of RIP1, RIP3 and MLKL from DFP-induced SE (Specific Aim 2); and (4) the Barnes maze, to test rats’ spatial learning and memory two one months after DFP injection, and to correlate findings with the numbers of remaining normal neurons in dorsal hippocampus (Specific Aim 4). We will analyze the data with multi-factor ANOVA and post-hoc t-tests, using pooled standard deviations and α = 0.05. We will determine if inhibition of RIP1 is neuroprotective and if optimal neuroprotection is provided by both 7-Cl-O-nec-1 and MK-801 given together, which, if true, should influence the standard of care following organophosphate exposure.