A new report in PNAS, Probing synaptic vesicle fusion by altering mechanical properties of the neuronal surface membrane, from Chuck Stevens’s lab, raises a serious concern about using styryl dyes to study release probability of synapses. Styryl dyes, such as FM 1-43, partition into cell membranes and have been commonly used to measure synaptic release of vesicles in culture and brain slice. The protocol is simple, bathe the neurons in dye, electrically stimulate to cause massive synaptic release and then dye uptake via vesicular endocytosis, wash off the dye, then observe the rate of destaining of the synapses following electrical stimulation. This rate is directly related to the number of vesicle fusions during the final stimulation period. There is just one problem, Zhu and Stevens report that the presence of the dye reduces the release probability of the synapse.
They observed this by taking the FM dye measurements in neurons that expressed synaptophysin-pHluorin. The fluoresence increase from the pHluorins was reduced in a [dye] dependent manner. A 15uM concentration of dye (not atypical for published experiments) reduced the pHluorin signal by 40%. The dye had no effect on the calcium levels in the presynaptic terminals, indicating it was potentially due to an increased energetic cost of forming a fusion pore. Chuck then weaves together some basic principles with this data to make an estimate of the fusion pore size.
While this paper may seem to cover a minor technical point, FM dyes have been used to make numerous inferences about presynaptic release properties, modes of vesicle recycling, the locus of LTP expression, and other basics of synaptic physiology. The thing that’s bugging me is if this effect is as prominent as advertised, how did people not notice the change in release probability with electrophysiological techniques?