Recruitment of release sites underlies chemical presynaptic potentiation at hippocampal mossy fiber boutons

Abstract

Synaptic plasticity is a cellular model for learning and memory. However, the expression mechanisms underlying presynaptic forms of plasticity are not well understood. Here, we investigate functional and structural correlates of presynaptic potentiation at large hippocampal mossy fiber boutons induced by the adenylyl cyclase activator forskolin. We performed 2-photon imaging of the genetically encoded glutamate sensor iGluu that revealed an increase in the surface area used for glutamate release at potentiated terminals. Time-gated stimulated emission depletion microscopy revealed no change in the coupling distance between P/Q-type calcium channels and release sites mapped by Munc13-1 cluster position. Finally, by high-pressure freezing and transmission electron microscopy analysis, we found a fast remodeling of synaptic ultrastructure at potentiated boutons: Synaptic vesicles dispersed in the terminal and accumulated at the active zones, while active zone density and synaptic complexity increased. We suggest that these rapid and early structural rearrangements might enable long-term increase in synaptic strength.

Fig 1. Two-photon imaging of single hippocampal mossy fiber bouton (hMFB) iGlu_u transients.

(A) Fluorescent image of an organotypic hippocampal slice culture 3 wk after transfection with the genetically encoded glutamate sensor iGlu_u in dentate gyrus (DG) granule cells. The square shows the region in (B–D). DG and CA3 are outlined by overlay. sl, stratum lucidum. (B) iGlu_u fluorescent signal acquired by 2-photon imaging in stratum lucidum (average of 15 frames). (C) Image of nonspecific autofluorescence with emission > 600 nm. (D) Composite of (B) and (C). The red rectangle marks the recorded area of the hMFB shown in (E). Note the position of the stimulation electrode indicated by the drawing. sp, stratum pyramidale. (E) High-resolution image illustrating the hMFB shown in (B) and (D) immediately before the high-speed imaging recordings. A hand-drawn green curve contours the bouton. (F–H) Single frames of the same hMFB showing ΔF/F signals at rest (F) and at the peak response after the first (G) and second (H) electrical stimulation in control conditions. The green line in (F–H) contours the hMFB silhouette shown in (E). Colored boxes represent pixels for which the intensity plots are shown in (I). (I) Plot representing dynamic ΔF/F fluorescent signals for each pixel in (F–H). Colored traces represent signals from pixels in colored boxes in (F–H). (J) Scheme illustrating the 2-photon laser scanning pattern with mean spatiotemporal resolution characteristics. (K) High-resolution image of a hMFB used for high-frequency stimulation experiments. The bouton contour was manually outlined by a green line. (L and M) Single frames of the hMFB illustrated in (K) showing ΔF/F signals at the peak response after the first (L) and sixth (M) electrical stimulation in control conditions. The green line shows the hMFB location. Contoured pixels represent active area. Note elevated pixel intensity and active area after 50-Hz stimulation. (N) The trace represents cumulative response dynamics for the given hMFB example in (K–M). The arrows indicate time points taken for illustration in (L) and (M). Note the almost 4-fold increase in cumulative ΔF/F signal after the last stimulation compared to the first, which illustrates the operating range of the iGlu_u sensor. El. stim., electrical stimulation. The data underlying this figure can be found at doi: 10.5281/zenodo.4498214.

Fig 2. Forskolin increases the presynaptic surface area of glutamate release and the spatial synchronization of glutamate release within hMFBs.

(A and B) Example images illustrating the spatial distribution of ΔF/F signals for 2 different hMFBs in control conditions (A) and in the presence of forskolin (B). The manually drawn green curve contours the profile of the recorded hMFB, based on the high-resolution image acquired before the stimulation experiment. The ΔF/F signals are taken at the peak responses to a first electrical stimulation (the time point is indicated by a black arrow in (C, F, J, L, N, and O). Suprathreshold pixels (pixels with ΔF/F intensities more than 3 × SD of the baseline signal, i.e., 50 ms before the stimulation) are contoured with a black line and represent the active area. Note the larger fraction of red pixels in the presence of forskolin (B) at equal intensities. (C) Example traces representing active area (the area of suprathreshold pixels) dynamics for hMFBs under control conditions (blue) and in the presence of forskolin (red). (D) Bar graph showing the active area at peak of response to the first stimulation normalized to the hMFB area. Note forskolin-mediated increase of hMFB active area. (E) Forskolin does not change the virtual bouton diameter (diameter of a circle with area equal to the area of the recorded bouton) of hMFBs. The bouton area was calculated using high-resolution images obtained before high-speed recordings. (F) Traces of cumulative intensities (spatial integral of suprathreshold pixels). The signal decay after the second stimulation is fitted with a monoexponential curve (thick lines) to identify the tau of decay (τ). (G–I). Bar graphs indicating the significant increase in cumulative amplitude in the presence of forskolin (maximal response to the first stimulation) (G), the decrease in the cumulative PPR (H), and the unchanged tau of decay of the cumulative intensities (I). (J) Traces of maximal ΔF/F values for suprathreshold pixels. (K) Bar graph showing that forskolin does not affect the maximal amplitude. (L) Traces of mean ΔF/F for suprathreshold pixels. (M) Bar graph showing that forskolin does not affect the mean amplitude. (N and O) Example traces representing informational entropy (N) and non-triviality (O) (see definitions in Methods) calculated for 2D patterns of ΔF/F spatial distributions at each time point for different hMFBs under control conditions (blue traces) and in the presence of forskolin (red traces). (P and Q) Bar graphs showing significantly decreased amplitudes of entropy (P) and non-triviality (Q) at the peak response to the fist stimulation. ampl., amplitude; CTRL, control; El. stim., electrical stimulation; FSK, forskolin; hMFB, hippocampal mossy fiber bouton; MFB, hippocampal mossy fiber bouton; MW, Mann–Whitney U test; MW-t, Mann–Whitney U test; PPR, paired-pulse ratio; ut-t, unpaired t test. The data underlying this figure can be found at doi: 10.5281/zenodo.4498214.

Fig 3. Coupling distance between Cav2.1 and Munc13-1 in the CA3 stratum lucidum is unchanged in control versus forskolin.

(A) Example scan in ZnT3-positive area of the CA3 stratum lucidum in 100-μm-thick hippocampal slices: confocal scan (top), raw gSTED scan (middle), and deconvolved gSTED scan (bottom). Staining for Cav2.1 (green), Munc13-1 (magenta), and Homer1 (cyan). (B) Example of an analyzed synapse: The distance between Cav2.1 (green) and Munc13-1 (magenta) was measured only if they were in close proximity to a Homer1-positive spot (cyan). Line profiles were plotted at the dotted line (top), drawn through the intensity maxima of the Cav2.1 and Munc13-1 signals (arrowheads). The distance was calculated between the intensity maxima of the Cav2.1 and Munc13-1 signals, shown in the example normalized intensity plots for control (middle) and forskolin-treated (bottom). (C) The distribution of measured distances between Cav2.1 and Munc13-1 in the CA3 stratum lucidum is unchanged in control versus forskolin-treated (p = 0.81, 2-sample Kolmogorov–Smirnov test). Frequency distribution (left y-axis, bars) and cumulative frequency (right y-axis, lines) with a bin size of 20 nm, for control (blue) and forskolin-treated (red). (D) The mean distance between Cav2.1 and Munc13-1 in the CA3 stratum lucidum is unchanged in control versus forskolin-treated. Scatter plot from all measured synapses: distances (nm) for CA3 control in blue (n = 584 synapses from 11 animals) and CA3 forskolin-treated in red (n = 525 synapses from 11 animals). Bar graphs show mean values ± SEM. Significance tested with Mann–Whitney U test (p = 0.62). CS, cumulative frequency; CTRL, control; FD, frequency distribution; FSK, forskolin; gSTED, time-gated stimulated emission depletion; KS, Kolmogorov–Smirnov test; MW, Mann–Whitney U test. The data underlying this figure can be found at doi: 10.5281/zenodo.4498214.

Fig 4. Three-dimensional EM analysis reveals an increase in presynaptic complexity and AZ density in forskolin-treated cryo-fixed acute slices.

(A) EM image of the stratum lucidum of the hippocampal CA3 region. Mossy fiber axon bundles (mf) are visible in the left panel. In the central panel, large presynaptic terminals contacting multiple spine heads (sp) are visible. The right panel shows a high-magnification image of a single AZ; this is a magnification of the spine marked in red (sp) in the middle panel. (B) Partial 3D reconstruction computed from manually segmented serial images of hMFBs in control conditions or after forskolin treatment. Presynaptic membrane is green, postsynaptic membrane is light blue, synaptic vesicles are yellow, AZs and docked or tethered vesicles are blue (control) or red (forskolin). (C) Bar graph indicating the quantification of bouton complexity (perimeter/area) obtained from images like the middle image of (A); bouton complexity was larger in forskolin-treated terminals (p = 0.0001, unpaired t test). (D) Bar graph indicating the quantification of AZ density (AZ/μm³) obtained from 3D reconstructions like those in (B); AZ density was larger in forskolin-treated terminals (p = 0.0035, Mann–Whitney U test). (E) Bar graph indicating the quantification of presynaptic area (μm²) obtained from images like the middle image of (A); presynaptic area was unchanged in forskolin-treated terminals when compared to controls (p = 0.07, unpaired t test). In all graphs, scatter points indicate individual boutons, n = 22 boutons for control and 20 boutons for forskolin-treated slices from 4 animals. Values represent mean ± SEM. AZ, active zone; CTRL, control; EM, electron microscopy; FSK, forskolin; hMFB, hippocampal mossy fiber bouton; MW, Mann–Whitney U test; ut-t, unpaired t test. The data underlying this figure can be found at doi: 10.5281/zenodo.4498214.

Fig 5. SVs disperse upon forskolin-induced presynaptic potentiation in cryo-fixed acute slices.

(A) Partial 3D reconstruction of hippocampal mossy fiber boutons in control conditions or after forskolin treatment. Presynaptic membrane is green, postsynaptic membrane is light blue, and SVs are blue (control) or red (forskolin). (B) Bar graphs indicating the quantification of SV density per cubic micron of reconstructed volume; SV density was comparable in forskolin-treated and control terminals (p = 0.5639, unpaired t test). (C) Bar graphs indicating the quantification of SV distance from other SVs normalized by the volume of the reconstruction (nm/μm³); distance between vesicles was increased in forskolin-treated terminals (p = 0.0050, Mann–Whitney U test). (D) Bar graphs indicating the quantification of MNND between vesicles (nm); MNND was comparable in forskolin-treated and control terminals (p = 0.1946, Mann–Whitney U test). In all graphs, scatter points indicate individual boutons, n = 22 boutons for control and 20 boutons for forskolin-treated slices from 4 animals. Values represent mean ± SEM. CTRL, control; FSK, forskolin; MNND, mean nearest neighbor distance; MW, Mann–Whitney U test; SV, synaptic vesicle; ut-t, unpaired t test. The data underlying this figure can be found at doi: 10.5281/zenodo.4498214.

Fig 6. Docked vesicle density increases upon forskolin-induced potentiation.

(A) Two-dimensional electron microscopy image from a high-pressure frozen mossy fiber AZ showing docked (light blue) and tethered (blue) SVs. (B) Three-dimensional reconstruction of mossy fiber AZs from acute slices cryo-fixed in control conditions or after forskolin treatment. Top panels show the xz views, and bottom panels the xy views. (C) Bar graph indicating the quantification of AZ area (μm²) for control and forskolin-treated boutons. (D–F) Bar graphs indicating the quantification of docked vesicle density in the whole bouton (docked SV/μm³) (D), docked vesicle density per square micron of AZ (docked SV/μm²) (E), and tethered vesicle density per square micron of AZ (tethered SV/μm²) (F) in control and forskolin-treated boutons. (G) Bar graph indicating the quantification of the putative readily releasable pool, measured as docked and tethered vesicle density per square micron of AZ (docked and tethered SV/μm²) in control and forskolin-treated boutons. Scatter points indicate the mean value for each individual bouton from 4 animals. Values represent mean ± SEM. AZ, active zone; CTRL, control; FSK, forskolin; MW, Mann–Whitney U test; SV, synaptic vesicle; ut-t, unpaired t test. The data underlying this figure can be found at doi: 10.5281/zenodo.4498214.