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

Abstract

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.

Keywords: long-term potentiation; nanoscale; synaptic plasticity; ultrastructure.

Fig. 1.

The density of docked vesicles at presynaptic active zones increased following LTP. Virtual images and surface models of docked and nondocked vesicles within 45 nm, which are tethered by filaments (yellow arrows) extending from the presynaptic membrane. The postsynaptic membrane is marked by dotted lines (cyan). (Scale bar in E is 50 nm for all EM virtual images. The models are to the same scale but are slightly rotated for ease of visualization, and the white 50-nm scale bar in D is for D and H, while the white 50-nm scale bar in I is for I–L.) (A–D) Control synapses: Virtual images of docked (A and B, purple arrows) and nondocked (C, blue arrowhead) synaptic vesicles at the presynaptic membrane. Virtual section thickness is 0.35 nm. (D) Representative 3D surface model of a control active zone showing one docked vesicle (purple), four nondocked vesicles (blue), and associated tethering filaments (yellow). (E–H) LTP synapses: Virtual images of docked (A and B, purple arrows) and nondocked (C, blue arrowhead) synaptic vesicles at the presynaptic membrane. Virtual section thickness is 0.27 nm. (H) Representative 3D surface model of a control active zone showing one docked vesicle (purple), four nondocked vesicles (blue), and associated tethering filaments (yellow). (I–L) Representative surface models of presynaptic membranes (silver) superimposed with projections from tightly docked vesicles (purple) at (I) control and (K) LTP synapses, or loosely docked (dark blue) and nondocked (light blue) vesicles at (J) control and (L) LTP synapses. An unbiased sampling density was computed per 0.01 µm² of presynaptic membrane surface area by counting all vesicles having projections contained within the rectangle or touching the green inclusion lines, and not counting vesicles touching the red exclusion lines. (M) Synaptic vesicle projection densities (control: blue, n = 22 synapses; LTP: red, 19 synapses). The density of docked vesicles was greater for LTP than control synapses (KS test, P = 0.02). The densities of loosely docked and nondocked vesicles were not significantly different between LTP and control synapses (KS test, P values listed). (N) Cumulative frequency plots of distances from loosely docked synaptic vesicles to the presynaptic membrane (SV–PM, KS test, P = 0.59) and from nondocked synaptic vesicles to the presynaptic membrane (SV–PM, KS test, P = 0.56). (Inset: red arrow shows how the distance from SV to the PM was measured.)

Fig. 2.

Vesicle contact area and filament occupancy of synaptic membranes. (A and B) Three-dimensional models of control (A) and LTP (B) synapses illustrate multiple filaments at the presynaptic membrane (yellow), in the synaptic cleft (green), and at the cytosolic face of the postsynaptic membrane (red). (The slightly rotated 50-nm scale bar in A is for A–F.) (C and D) Three-dimensional synapse models of presynaptic membranes from A and B rotated 90° to reveal filament (yellow) and vesicle contact areas (dark blue) for control (C) and LTP (D) synapses. (E and F) Three-dimensional synapse models of postsynaptic membranes from A and B rotated 90° to reveal postsynaptic filaments (red) for control (E) and LTP (F) synapses. (G and H) Scatter plots with box plots (G) and cumulative frequency plots (H) of docked vesicle contact areas with the presynaptic membrane from control (blue; n = 96 vesicles) and LTP (red; n = 111 vesicles) synapses. The mean contact areas (control, 240 ± 160 nm²; LTP, 210 ± 160 nm²) and overall distributions did not differ (t test, P = 0.31; KS test, P = 0.14). (I and J) Plots for pairs of relative filament occupancy (area ratio) on postsynaptic and presynaptic membranes of control (I; n = 22) and LTP (J; n = 19) synapses. Area ratio was calculated by dividing the total filament contact area by the imaged presynaptic or postsynaptic membrane area of each synapse. The contact area of the docked vesicles was subtracted from the total imaged presynaptic membrane area. The relative occupancy (area ratio) of filaments on the postsynaptic membrane was greater than on the presynaptic membrane for LTP synapses (J: 0.36 ± 0.12, t test, P = 3.3 × 10⁻⁵), but not control synapses (I: 0.31 ± 0.19, t test, P = 0.052). (K) The area ratio of filaments on the postsynaptic membrane was greater with LTP (t test, P = 0.021), but was not different on the presynaptic membrane (t test, P = 0.39).

Fig. 3.

Shortened distances between tethering filaments and presynaptic membranes of the active zone following LTP. Virtual sections and surface models of tightly docked vesicles in control (A and A′, purple) and LTP (B and B′, purple) conditions and nondocked vesicles in control (C and C′, blue) and LTP (D and D′, blue) conditions. The virtual images are 0.35 nm thick for the control synapses (A and C) and 0.27 nm thick for the LTP synapses (B and D). Tethering filaments have matching arrows in the virtual sections and surface models (yellow). (Scale bar: 50 nm; in A for A–D and in A′ for the 3D models with slight rotation.) (E) Three distances were measured separately for every tethering filament on tightly docked and loosely or nondocked vesicles that were incomplete or complete in these EMT samples (see Table 1 for n values in each category). Distance was measured between the filament attachment site on the vesicle (Fil_VA) projected onto the presynaptic membrane (PM) (closed circle and black arrow in the Insets). Distance was measured between the Fil_VA and its filament attachment site on the presynaptic membrane (Fil_PA, closed triangle and black arrow in the Insets). The distance between these projections along the active zone membrane was calculated assuming a right triangle (PM–Fil_PA, open square and dotted line in the Insets). For tightly docked vesicles, all three values were shorter after LTP relative to control stimulation (Fil_VA–PM, control = 8.5 ± 4.1 nm and LTP = 7.3 ± 3.5 nm, KS test, P = 0.0001; Fil_VA–Fil_PA, control = 11 ± 5.5 nm and LTP = 9.5 ± 4.5 nm, KS test, P = 0.0001, and PM–Fil_PA, control = 6.6 ± 4.9 nm and LTP = 5.5 ± 3.9 nm, KS test, P = 0.017). For loosely docked or nondocked vesicles, the Fil_VA–PM and Fil_VA–Fil_PA distances were both shortened after LTP compared to control conditions (Fil_VA–PM: control, 21 ± 13 nm, and LTP, 19 ± 12 nm; KS test, P = 0.042; and Fil_VA–PM_PA: control, 26 ± 15 nm, and LTP, 23 ± 13 nm; KS test, P = 0.039); however, the calculated PM–Fil_PA was not significantly different between conditions (13 ± 9.6 and 12 ± 7.4 nm; KS test, P = 0.46). (F) Tethering filament distances were averaged for every tightly docked vesicle that was complete in the EMT series and compared to the contact area of the docked vesicle with the presynaptic membrane. These distances were very slightly shorter at larger contact areas for both control and LTP conditions (see r values on graphs). The average Fil_VA–PM, Fil_VA–Fil_PA, and PM–Fil_PA distances were significantly shorter across the full range of contact areas in the LTP than control synapses (ANCOVA results are on the graphs; for Fil_VA–PM, F = 13, P = 0.0004, for Fil_VA–Fil_PA, F = 14, P = 0.0003, and for PM–Fil_PA, F = 4, P = 0.04). (G) Tethering filament distances were averaged for every loosely docked or nondocked vesicle that was complete in the EMT series and compared to the synaptic vesicle’s distance from the presynaptic membrane (SV–PM). The average distances of Fil_VA–PM, Fil_VA–Fil_PA, and PM–Fil_PA were highly positively correlated with the SV–PM distance (for control, Fil_VA–PM, P = 9 × 10⁻⁴², Fil_VA–Fil_PA, P = 9 × 10⁻³¹, PM–Fil_PA, P = 9 × 10⁻⁷; and for LTP, Fil_VA–PM, P = 9 × 10⁻²³, Fil_VA–Fil_PA, P = 2 × 10⁻¹⁹, and PM–Fil_PA, P = 2 × 10⁻⁵). ANCOVA revealed Fil_VA–PM distances were shorter after LTP (F = 10, P = 0.003) as was the Fil_VA–Fil_PA (F = 7, P = 0.01). However, the PM–Fil_PA was not significantly reduced after LTP (F = 1, P = 0.3).

Fig. 4.

Modeling effects of LTP on synaptic vesicles and tethering filaments. Tightly docked and loosely or nondocked synaptic vesicles (SV; purple) have filaments (gold) tethering them to the presynaptic membrane (PM; gray). By 2 h following the induction of LTP, the density of tightly docked vesicles nearly doubles. For all three vesicle locations, the attachment sites are displaced lower (vertical dotted arrows) and the filaments become shorter (angled dotted arrows). For the tightly docked vesicles, the attachments sites on the presynaptic membrane shift closer to the vesicle (horizontal arrowheads).