Confocal imaging and tracking of the exocytotic routes for D ‐serine‐mediated gliotransmission

D ‐Serine is an astrocyte‐derived regulator for N ‐methyl‐ D ‐aspartate receptors, but the intracellular routes of its trafficking are still largely unknown. Here, we combined confocal microscopy with colocalization quantification to track the astrocytic organelles that store D ‐serine. We report that D ‐serine colocalizes with the transfected eGFP‐synaptobrevin/VAMP2 and eGFP‐cellubrevin/VAMP3, two v‐SNAREs of the regulated secretory pathway. No significant colocalization was found with markers of the endosomal sorting and recycling system: EEA1, eGFP‐endobrevin/VAMP8, eGFP‐TI‐VAMP/VAMP7, LAMP1, and CD63. Blockade of vesicular budding with colchicine shows that secretory vesicles import D ‐serine downstream to the Golgi apparatus. Finally, treatment of astrocytes with the Ca 2+ ‐ionophore A23187, glutamate agonists, or bradykinin trigger translocation of synaptobrevin/VAMP2 to the plasma membrane with a concomitant disappearance of D ‐serine from the regulated secretory pathway. Our results provide morphological evidence for a vesicular storage of D ‐serine in the regulated secretory pathway and the possible recruitment of these stores by Ca 2+ mobilization to release D ‐serine. © 2008 Wiley‐Liss, Inc.