Project Summary Intravenous lipid emulsions (ILEs) are important therapies. Though designed for parenteral nutrition, they were discovered to be an antidote for local anesthetic systemic toxicity (LAST). With rising intravascular concentrations of local anesthetics (LAs), which canonically inhibit voltage-gated sodium channels (Nav), symptoms of central nervous system (CNS) toxicity progress to seizures, and cardiac arrest may ensue. LAST is a major clinical challenge, and the identification of ILEs as treatment was a breakthrough that has made use of LAs safer. Despite this important role, the molecular mechanisms by which ILEs treat LAST are not fully understood. Furthermore, there is a paucity of studies examining whether Intralipid®, the emulsion recommended in medical guidelines that is composed of long-chain triglycerides from soybean oil, is the best choice. The most simplistic model for the effects of ILEs is that they form an intravascular compartment to partition LAs and lower the effect-site concentration, but the degree to which concentrations fall in the aqueous phase is debated. A competing hypothesis with strong evidence in cardiac models proposes ILEs serve as a fuel via β-oxidation of triglycerides (TGs), overcoming mitochondrial dysfunction caused by LAs. This mechanism has been dismissed to explain reversal of CNS dysfunction because of the dogmatic belief that neurons rely on glucose metabolism. Additionally, neurotoxicity due to LAs has been attributed, with limited evidence, to preferential block of inhibitory neurons, but important effects in the CNS are unexplained by this hypothesis. My preliminary in vitro data shows that neurons can use Intralipid® to sustain synaptic function in the absence of other fuels, supporting the metabolic hypothesis of ILEs for LAST in the CNS. This research proposal will test the hypothesis that ILEs reverse neurotoxicity by overcoming LA-induced mitochondrial dysfunction. The experimental plan will systematically investigate the capacity for neurons to metabolize components of ILEs. In doing so, I will comprehensively investigate the suitability of lipids to fuel synaptic function. In Aim 1, I will use my developing expertise in optical imaging of cultured neurons expressing genetically-encoded biosensors to compare lipids by their ability to sustain synaptic vesicle recycling and produce ATP when deprived of glucose. Lipids will be tested as emulsions of both long- and medium-chain TGs and free fatty acids of different lengths and saturation. In Aim 2, a metabolically optimized emulsion will be compared in culture to Intralipid® in its ability to reverse LA-induced synaptic dysfunction. In Aim 3, the optimized emulsion will be compared to Intralipid® in mice, quantifying effects on seizures with widefield calcium imaging, local field potentials, and autofluorescence flavin imaging of metabolic activity. My five-year research and career proposals capitalize on my excellent mentors and inst...