The transfer of information at a synapse requires release of neurotransmitter from a presynaptic terminal and the subsequent activation of postsynaptic receptors. All synapses show both evoked (calcium-dependent) and spontaneous (calcium-independent) forms of neurotransmitter release 1
. Evoked release is tightly coupled to the action potential and generates a postsynaptic response with sub-millisecond delay 2
. At rest, in the absence of any activity, the machinery responsible for the fusion of neurotransmitter-filled vesicles at a synapse has a very low release probability, but is not zero. As a result, spontaneous fusion events occur stochastically, but at a very low rate, estimated to be around 1-2 vesicle per minute 3,4
. The consensus view is that membrane depolarization (by action potentials or otherwise) increases the probability of vesicle fusion allowing the synapse to switch from a negligible rate of spontaneous release to a rapid synchronized form of release. Recently, a debate has ensued regarding the origin of each mode of release 5,6
. Do vesicles that release neurotransmitter spontaneously draw from the same pool as those released in response to neuronal activity? Related to this question, is spontaneous (or constitutive) release an important feature of synaptic transmission? These basic, yet unresolved questions are crucial to understanding information transfer at the synapse and probe some of the basic assumptions of quantal theory 1
. To advance our knowledge in this area it is important to first establish the identity of presynaptic vesicle pools and understand how they are mobilized within a terminal.
Presynaptic neurotransmitter-filled vesicles are organized into distinct pools, with different kinetics of release and subcellular localization 7
. In general, vesicles are thought to be arranged into three main pools: a rapidly releasable pool (RRP), a reserve pool and a resting pool. The sizes of each pool, their location within the synapse, as well as the modes in which they are mobilized are still a matter of debate 7
. It is generally thought that the RRP constitutes those vesicles closest to the plasma membrane, docked at the active zone and ready for immediate release 8
. The reserve pool serves to refill the empty release sites at the RRP and acts as a source of vesicles, which the bouton can draw on to continue releasing neurotransmitter after the RRP has been used up. These two pools are collectively known as the recycling pool and are mobilized by membrane depolarization in a calcium-dependent manner. Finally, there is a group of vesicles known as the resting pool, which are refractory to release in response to electrical activity 3,9
. The function of this pool of vesicles has remained largely unknown 10
Spontaneous events, known as miniature postsynaptic events or minis, are postsynaptic measures that reflect the release of neurotransmitter released from a single vesicle (or quanta) and were used to formulate the quantal theory of neurotransmitter release 1
. From a presynaptic perspective, dyes that label membranes, such as FM1-43, have shown that spontaneous vesicle exo- and endocytosis also occur at the synapse and concluded that these events correspond to the postsynaptic mini 3,11
. One widely accepted assumption is that vesicles undergoing both spontaneous and activity-dependent fusion use the same set of vesicles and the same fusion machinery. More recent studies have proposed that these two modes of release may draw from different pools 5,12
. To date, conflicting reports have found evidence both for and against two separate pools of vesicles, leaving the question unresolved 6,11
. Here, we develop and implement a novel technique to label vesicles in live neurons. Using this method, we show the existence of two independent pools of vesicles: one that fuses spontaneously with the plasma membrane and another that can only be mobilized by neuronal activity. In addition, we used the presynaptic probe synaptophysin-pHluorin (sypHy) to establish the identity of the spontaneous vesicle pool. We find that spontaneously-released vesicles are mobilized from a ‘resting pool’ that was originally described as an activity-independent set of vesicles that do not participate in vesicle cycling. Our data not only provides further evidence for the existence of a functionally heterogeneous population of vesicles in presynaptic terminals, but also establishes a role for the previously ill-defined resting pool.