Imaging FM Dyes in Brain Slices

INTRODUCTIONThis protocol describes a method for monitoring the activity-dependent loading and unloading of synaptic vesicles with a fluorescent probe in intact or semi-intact neuronal systems. It is a fairly general method that could be applied to a number of biological preparations other than nervous systems. So far, the method has been applied to Caenorhabditis elegans, Xenopus tadpoles, lamprey spinal cord, fly brain, rat brain, and mouse brain. The method relies on the chemical properties of styryl pyridinium probes, such as FM1-43, that are somewhat water-soluble but preferentially partition into lipid environments. The probe is taken up by endocytosis and only fluoresces once in the membrane. Stimulation of exocytosis releases the probe into the extracellular space, with a consequent decline in fluorescence, allowing the time course of release to be followed. In intact systems such as brain or brain slices, FM1-43 adsorbs to the extracellular surface of the plasma membrane and is resistant to removal by washing. This gives rise to an intense fluorescence signal associated with the extracellular membrane, which obscures the weaker signal associated with vesicular uptake. FM1-43 and its congeners can be removed by using a modified cyclodextrin (Advasep-7) that in effect serves as a soluble high-affinity scavenger for the probe, allowing it to be washed out of the preparation. Cyclodextrins are water-soluble, doughnut-shaped molecules with hydrophobic holes of the right size to accommodate FM1-43, forming what is called an "inclusion" complex.