Enhanced cycling of presynaptic vesicles during long-term potentiation in rat hippocampus

Abstract

Long-term potentiation (LTP) is a widely studied form of synaptic plasticity engaged during learning and memory. Here the ultrastructural evidence is reviewed that supports an elevated and sustained increase in the probability of vesicle release and recycling during LTP. In hippocampal area CA1, small dense-core vesicles and tethered synaptic vesicles are recruited to presynaptic boutons enlarging active zones. By 2 h during LTP, there is a sustained loss of vesicles, especially in presynaptic boutons containing mitochondria and clathrin-coated pits. This decrease in vesicles accompanies an enlargement of the presynaptic bouton, suggesting they supply membrane needed for the enlarged bouton surface area. The spatial relationship of vesicles to the active zone varies with functional status. Tightly docked vesicles contact the presynaptic membrane and are primed for release of neurotransmitter upon the next action potential. Loosely docked vesicles are located within 8 nm of the presynaptic membrane. Non-docked vesicles comprise recycling and reserve pools. Vesicles are tethered to the active zone via filaments composed of molecules engaged in docking and release processes. Electron tomography reveals clustering of docked vesicles at higher local densities in active zones after LTP. Furthermore, the tethering filaments on vesicles at the active zone are shorter, and their attachment sites are shifted closer to the active zone. These changes suggest more vesicles are docked, primed and ready for release. The findings provide strong ultrastructural evidence for a long-lasting increase in release probability following LTP.

Keywords: axon; dendrite; nanoscale; spines synapse; synaptic plasticity; ultrastructure.

Figure 1. Homeostatic synaptic plasticity during LTP in the young adult rat hippocampus

A, positioning of stimulating electrodes and sample locations where serial sections were obtained for electron microscopy (EM) from the control (blue) and LTP (red) sites. The stimulating electrode that induced LTP was alternated between the CA3 (left) and the subicular (right) side of the recording electrode (black arrow). B, LTP was induced by theta‐burst stimulation (TBS; 8 trains at 30 s intervals of 10 bursts per train at 5 Hz with 4 pulses in each burst at 100 Hz), and the control side received test pulses at the same rate as LTP test pulses of 1 per 2 min over the course of the experiment. C, illustration of the excellent ultrastructure from an adult hippocampal slice using our rapid microwave‐enhanced fixation protocol (Jensen & Harris, 1989). This example is from a P61 rat hippocampus in the 30 min control condition. Red lines indicate example postsynaptic densities (PSD). D, the average size of PSD areas of synapses enlarged during LTP; (E) while the increase in small‐spine density seen under control conditions was absent during LTP. F, consequently, the total PSD area after LTP induction equalled that in control conditions. G, together these findings resulted in a homeostatic balance in total synaptic weight per unit length of dendrite. A–D, F and G, are adapted from Bourne & Harris (2011); E is adapted from Bell et al. (2014). *P < 0.05; ***P < 0.001.

Figure 2. CA3 axons that synapse in s. radiatum of hippocampal area CA1

A, central EM and 3D reconstructions of these axons illustrating their non‐parallel trajectories that give rise to stimulation of independent synapses at a separation between the electrodes greater than 200 µm using the protocol shown in Fig. 1. B, illustration showing that only 50% of the presynaptic boutons along these axons contain mitochondria (dark grey structures) in the perfusion‐fixed hippocampus, in vivo. (A and B are adapted from Shepherd & Harris, 1998). C–F, reconstructed axons from the control (Con) conditions (C, E) and 30 min (D) or 2 h (F) after induction of LTP in the same slice. Axon, pale blue; vesicles, green spheres; mitochondria (mito), fuchsia; PSDs, red. Adapted from Bourne et al. (2013).

Figure 3. The stalled spine outgrowth results in fewer single synaptic boutons (SSBs)

Aa–Ca, EM and b, 3D reconstructions of SSB A, multisynaptic bouton (MSB) B, and non‐synaptic bouton (NSB) (C). ax, axon; sp, spine. D, central EM and E, sampling brick across serial EM volume to identify and include boutons contained within or touching the green surfaces while excluding those touching the red surface. F, brick analyses revealed no differences at 30 min, but fewer SSBs at 2 h after induction of LTP (P < 0.05) with no significant changes in MSBs or NSBs. Scale bar in Ca is for Aa–Ca and cube in Cb is for Ab–Cb at 0.5 µm per side, 0.125 µm³.

Figure 4. Sustained decrease in number of presynaptic vesicles during LTP

A, EM and B, 3D reconstruction of a dendritic spine (sp, yellow) and presynaptic bouton (ax) with the docked vesicles (blue), vesicles in the pool (green), and PSD (red). C, average number of docked vesicles for each condition (black lines) with distribution from individual boutons (n = number of boutons in each condition). D, decrease in the number of docked vesicles per presynaptic bouton at 30 min after induction of LTP relative to control values (P < 0.01). E, average number of non‐docked vesicles (black lines) for each condition with distribution from individual boutons. F, by 2 h after LTP induction, the vesicle pool was significantly smaller relative to control (P < 0.01).

Figure 5. Vesicle drop during LTP is greatest in boutons containing a mitochondrion

A and B, representative vesicle composition in presynaptic boutons (A) containing mitochondria (dark blue) or (B) without mitochondria from the control and LTP conditions. C–F, both docked and non‐docked vesicles had greater drops across synapses of all PSD areas for boutons with mitochondria (C, D) versus no mitochondrion (E, F). Adapted from Smith et al. (2016).

Figure 6. The number of vesicles was substantially reduced in boutons containing clathrin coated pits (CCP)

A–D, EM and 3D reconstructions of presynaptic vesicles (green), CCPs (orange), PSDs (red) and spines (yellow) from control (A, B) and 2 h LTP (C, D) conditions. E and F, graphs of vesicle counts in presynaptic boutons with and without one or more CCPs. G and H, statistically significant reductions (*P < 0.05, **P < 0.01) in docked vesicles and vesicle pool numbers in boutons with (+CPP) or without (–CCP) one or more CCPs.

Figure 7. Small dense core vesicles and the recruitment of presynaptic vesicles to convert nascent zones to active zones during LTP

A, active zone (AZ, red) with docked (royal blue arrow), non‐docked (white arrow), and reserve pool (green arrow) vesicles, at a PSD (red). B and C, nascent zone (NZ, aqua) has a PSD but no presynaptic vesicles. D and E, 3D reconstructions of the synapse illustrated in A–C. F and G, 3D reconstructions of axons in s. radiatum of hippocampal area CA1 illustrating their composition of synaptic vesicles (light green spheres), dense core vesicles (DCV), mitochondria (mito), and multivesicular body (MVB). These reconstructions are from perfusion fixed brain and illustrate neighbouring single‐synaptic (SSB), multisynaptic (MSB), and non‐synaptic (NSB) boutons along single axons. H, EM illustrating synaptic vesicles that are tethered (green arrows) to (I) a dense core vesicle (DCV). I, DCV at the edge of an AZ. J DCVs are recruited from inter‐bouton regions to synaptic boutons at 5 min after TBS (n = number of synapses). K, plot of the number of DCVs that would be needed to convert a NZ to AZ by filling it with the tethered docked vesicles versus the number of NZs in the control or LTP conditions that would be fully converted to AZ if a DCV were to be recruited with tethered vesicles. L, NZ area decreases by 30 min after LTP induction. M, NZ size is re‐elevated by 2 h after LTP induction. N, NZ recovery at 2 h during LTP is greatest on spines with the largest AZ areas (Perf: in vivo controls). (Adapted from Bell et al., ; Harris et al., ; Sorra et al., .)

Figure 8. Effects of LTP on the local density of tethered and docked presynaptic vesicles

A–J, tightly docked (purple), loosely docked (turquoise) or non‐docked (light blue) vesicles with molecular tethering filaments (yellow). C and D, side view of three‐dimensional tomogram illustrates presynaptic membrane surface (silver) with tight and loose docked vesicles and tethering filaments. E–J, unbiased sampling frames to compute local cluster densities for tight (E, F) and loose or non‐docked (I, J) vesicles. K, the density of tightly docked vesicles, but not loosely or non‐docked vesicles, increases in local clusters after LTP. L–P, the length of the tethering filaments (yellow) for both tight (L, M) and loosely docked (N, P) vesicles was shortened at 2 h after LTP induction. (Adapted from Harris et al., ; Jung et al., .)