Supplementary Material for: NextGen Brain Microdialysis: Applying Modern Metabolomics Technology to the Analysis of Extracellular Fluid in the Central Nervous System C.-Y.Kao E.Anderzhanova J.M.Asara C.T.Wotjak C.W.Turck 2015 Microdialysis is a powerful method for in vivo neurochemical analyses. It allows fluid sampling in a dynamic manner in specific brain regions over an extended period of time. A particular focus has been the neurochemical analysis of extracellular fluids to explore central nervous system functions. Brain microdialysis recovers neurotransmitters, low-molecular-weight neuromodulators and neuropeptides of special interest when studying behavior and drug effects. Other small molecules, such as central metabolites, are typically not assessed despite their potential to yield important information related to brain metabolism and activity in selected brain regions. We have implemented a liquid chromatography online mass spectrometry metabolomics platform for an expanded analysis of mouse brain microdialysates. The method is sensitive and delivers information for a far greater number of analytes than commonly used electrochemical and fluorescent detection or biochemical assays. The metabolomics platform was applied to the analysis of microdialysates in a foot shock-induced mouse model of posttraumatic stress disorder (PTSD). The rich metabolite data information was then used to delineate affected prefrontal molecular pathways that reflect individual susceptibility for developing PTSD-like symptoms. We demonstrate that hypothesis-free metabolomics can be adapted to the analysis of microdialysates for the discovery of small molecules with functional significance.