Presynaptic calcium measurements using bulk loading of acetoxymethyl indicators

We describe a method for labeling presynaptic terminals in mammalian brain slices by focal application of calcium indicators conjugated with acetoxymethyl (AM) esters. A solution of membrane-permeant, AM-conjugated calcium indicator is focally applied to the transverse cerebellar brain slice and allowed to equilibrate throughout the parallel fiber tract. Fibers are then stimulated with an extracellular electrode and fluorescence transients are measured from a location several hundred micrometers from the loading site using a photodiode or photomultiplier tube. Considerations for selecting an appropriate indicator and determining the optimum loading conditions are discussed.