Shortened tethering filaments stabilize presynaptic vesicles in support of elevated release probability during LTP in rat hippocampus

Long-term potentiation (LTP) is a cellular mechanism of learning and memory that results in a sustained increase in the probability of vesicular release of neurotransmitter. However, previous work in hippocampal area CA1 of the adult rat revealed that the total number of vesicles per synapse decreases following LTP, seemingly inconsistent with the elevated release probability. Here, electron-microscopic tomography (EMT) was used to assess whether changes in vesicle density or structure of vesicle tethering filaments at the active zone might explain the enhanced release probability following LTP. The spatial relationship of vesicles to the active zone varies with functional status. Tightly docked vesicles contact the presynaptic membrane, have partially formed SNARE complexes, and are primed for release of neurotransmitter upon the next action potential. Loosely docked vesicles are located within 8 nm of the presynaptic membrane where SNARE complexes begin to form. Nondocked vesicles comprise recycling and reserve pools. Vesicles are tethered to the active zone via filaments composed of molecules engaged in docking and release processes. The density of tightly docked vesicles was increased 2 h following LTP compared to control stimulation, whereas the densities of loosely docked or nondocked vesicles congregating within 45 nm above the active zones were unchanged. The tethering filaments on all vesicles were shorter and their attachment sites shifted closer to the active zone. These findings suggest that tethering filaments stabilize more vesicles in the primed state. Such changes would facilitate the long-lasting increase in release probability following LTP.

Long-term potentiation (LTP) is the persistent strengthening of synapses after a brief high-frequency stimulation and is widely accepted as a cellular correlate of learning and memory (1, 2). Within minutes after the induction of LTP, new receptors are inserted into the postsynaptic membrane. The resulting increase in the excitatory postsynaptic potential is immediate and can persist for hours in vitro or days to months in vivo (1, 3–8). Quantal content is also increased soon after LTP induction and reflects an increase in the number of presynaptic vesicles that release neurotransmitter (9–13). This increase in release probability is sustained several hours following LTP (14), concurrent with postsynaptic growth and spine enlargement (15). One might expect that the enhanced probability of release would involve increasing the number of vesicles docked and primed for neurotransmitter release. However, 2 h after induction of LTP, the total number of both docked and nondocked vesicles per presynaptic bouton are markedly decreased relative to control stimulation (16). These findings raise the question of whether an altered structure of docking and priming molecules leads to local clustering of vesicles that would elevate the probability of release following LTP.The proteins that connect synaptic vesicles to the plasma membrane can be visualized as filaments with electron-microscopic tomography (EMT) connecting vesicles to the presynaptic active zone (17–24). Studies suggest that the SNARE complex begins to form when a vesicle and presynaptic membrane are within 8 nm of each other rendering them loosely docked (25–27). Vesicles are then drawn toward the active zone, and the SNARE complex bundle is fully formed when the vesicle is within 2 nm of the presynaptic membrane (26, 28). Tightly docked vesicles are defined as being in contact with the presynaptic membrane and correspond to primed vesicles that comprise the readily releasable pool (27). Recent studies have suggested that docked vesicles can oscillate between loosely and tightly docked states (27, 29), providing a target mechanism for synaptic plasticity.To address the question of how changes in vesicle proximity and tethering might enhance the probability of release, we used EMT, which enabled us to acquire high-resolution structural data from small volumes of presynaptic boutons that were enriched in synaptic vesicles. We targeted active zones of hippocampal synapses, comparing their structure 2 h after LTP induction to control stimulation. The vesicle density and tethering filament dimensions were unchanged for the loosely docked and nondocked vesicles. In contrast, the density of tightly docked vesicles was increased, their tethering filaments were shorter, and the filament attachment sites on the vesicles were positioned closer to the side of the vesicle membrane facing the presynaptic membrane. Such alterations could contribute to the sustained increase in the probability of neurotransmitter release following LTP.