Science goes through waves of innovation driven by Ca2+ influx normally triggers neurotransmitter release technical advances. One recent such advance was the from a presynaptic terminal only unreliably, with one discovery of FM dyes as a tool to measure presynaptic release event per five to ten Ca2+ signals (Dobrunz and activity by optical recordings (reviewed in Cochilla et al., Stevens, 1997), resulting in a low overall synaptic release 1999). FM1-43 and its cousins are lipophilic fluorescent probability. Whenever release is triggered, however, probesthatspontaneouslyinsertintomembraneswhere usually only a single synaptic vesicle undergoes exothey become more flourescent. Importantly, these dyes cytosis, although all of the vesicles in the RRP appear can be easily washed out but do not cross bilayers. As to be ready for fusion. The restriction of exocytosis to a result, when a cell surface is exposed to FM dyes and a single vesicle is probably not an active process, but subsequently washed with dye-free solution, only those simply caused by the low release probability of each membranesthathavebeenrecapturedandarenolonger individualvesicle. Thissuggeststhattheoverallsynaptic surface exposed retain the fluorescent dye. Reexorelease probability is the sum of the individual vesicular cytosis of these membranes then leads to release of the release probabilities. Consistent with this idea, the numdye. Since membrane-bound dye is much more fluoresber of vesicles in the RRP appears to be a major determicent than dye in aequous solution, treatments of cell nant of the synaptic release probability (Dobrunz and surfaces with FM dyes followed by washes provide a Stevens, 1997). sensitive way of monitoring exo-and endocytosis. This The RRP of synaptic vesicles is by definition the most approach has been particularly useful for studying synimportant vesicle pool for neurotransmitter release. aptic vesicle recycling in presynaptic nerve terminals. However, this pool constitutes only a tiny fraction of In the most recent application of this technological innothe total vesicle pool. The RRP is a component of the vation, Pyle et al.(2000) describe a new pathway in the recycling pool of vesicles, defined as all vesicles that can synaptic vesicle cycle using two FM dyes that differ be labeled with FM1-43 when synapses are stimulated in the speed with which they equilibrate between the extensively. After extensive stimulation, the RRP is delipophilic membrane and aqueous solution. Using this pleted and additional vesicles are recruited for exoingenious approach, Pyle and colleagues observed that cytosis from the so-called “reserve pool” to the active synaptic vesicles can be immediately reused after exozone. The RRP and the reserve pool together constitute cytosis, probably without ever leaving the active zone the recycling pool (Figure 1). The total recycling pool of the presynaptic plasma membrane. In an independent has been estimated at 21–25 vesicles/synapse in hippostudy, Stevens and Williams (2000) also identify a fast campal cultures, with 17–20 vesicles in the reserve mode of vesicle reuse during exocytosis based on stainpool (Murthy and Stevens, 1999). Presynaptic terminals ing and destaining with FM dyes. These observations thus contain a small number of active vesicles (the recyclarify several unresolved issues and give rise to a new, cling pool), of which a third is in the RRP. Since morphomore economical view of the synaptic vesicle cycle. logically the same synapses were shown to contain However, they also raise general questions of how many 200 synaptic vesicles (Schikorski and Stevens, 1997), different vesicle pathways are nested …