An Enzymatic-HPLC Assay to Monitor Endogenous d-Serine Release from Neuronal Cultures

d -Serine is a transmitter-like molecule that physiologically activates NMDA receptors in the brain. Although d -serine was thought to be exclusively released by astrocytes, we recently demonstrated endogenous d -serine release from neurons in cultures and slices. So far high-performance liquid chromatography (HPLC) has been the standard technique to monitor d -serine and other amino acids. This method employs pre-column derivatization with a chiral reagent to produce fluorescence derivatives that can be further separated on a reversed-phase column. Due to the close retention times of l -serine, l -glutamine, and d -serine, the quantification of low levels of endogenous d -serine synthesis and release from cell cultures and tissues can be challenging. We here describe an enzymatic treatment method to specifically destroy l -glutamine and l -serine by glutaminase and l -serine dehydratase enzymes, respectively, allowing accurate determination of nanomolar d -serine concentrations by subsequent HPLC analysis.