The overall goals of this grant application are to study potential mechanisms associated with regulation of neuronal transbilayer and lateral cholesterol domains and how cholesterol domains interact with calcium homeostasis in neuronal membranes of different age groups of mice. We have recently shown in synaptic plasma membranes (SPM) of young C57BL mice, that the two membrane leaflets are asymmetric in their fluidity and cholesterol distribution. Membrane cholesterol was also found to be located in exchangeable and non-exchangeable pools and membrane cholesterol domains could be altered by hydrolysis of sphingomyelin that also altered intracellular calcium. Preliminary data from out laboratory showed that the asymmetry of fluidity between the two SPM leaflets observed in 6 month old animals was significantly reduced in SPM of 28 month old animals and that the exofacial leaflet was more affected as compared to the cytofacial leaflet. Previous studies on membrane lipid structure in different age groups of animals have examined changes in the average fluidity of the membrane lipid structure, whereas other studies have not observed age differences. We propose that increasing age is associated with marked changes in membrane lipid structure but that such changes are occurring in specific lipid domains and changes in lipid domains contribute to modification of calcium homeostasis that has been previously shown to differ with increasing age. The proposed experiments will focus on SPM and synaptosomes of 4, 16, and 28 month old C57BL/NNia mice. The specific aims of this application are to: 1) determine transbilayer distribution of cholesterol in the SPM exofacial and cytofacial leaflets; 2) examine cholesterol lateral domains in SPM; 3) phospholipid distribution in SPM exofacial and cytofacial leaflets; 4) involvement of sphingomyelin in regulation of cholesterol domains; 5) brain sterol carrier proteins and regulation of cholesterol domains; and 6) cholesterol domains and presynaptic calcium. It is our working hypothesis that aging impairs the capacity of the membrane to regulate cholesterol domains and such changes in cholesterol domains modify neuronal calcium.