Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion

Abstract

Exo-endocytotic turnover of synaptic vesicles (SVs) at synapses between hippocampal neurons in culture was examined by electron microscopy (EM). We carried out photoconversion (PC) of the fluorescent endocytotic marker FM 1-43 by using 3,3'-diaminobenzidine to convert the dye signal into an electron-dense product. Electron-dense products were located almost exclusively in SVs, whose densities were bimodally distributed in two sharply demarcated populations, PC-positive (PC+) and PC-negative (PC-). The median densities of these populations did not vary with the proportion of vesicles stained within a presynaptic terminal (bouton). The proportion of PC+ SVs remained constant across consecutive thin sections of single boutons, but varied greatly from one bouton to another, indicating marked heterogeneity in exo-endocytotic activity. Our experiments indicated that only a minority of SVs were stained in most boutons after stimuli known to cause complete turnover of the functional vesicular pool. A direct spatial correlation was found between FM 1-43 fluorescent spots seen with light microscopy and PC+ boutons by EM. The correlation was clearer in isolated boutons than in clusters of boutons. Photoconversion in combination with FM dyes allows clarification of important aspects of vesicular traffic in central nervous system nerve terminals.

Figure 1

Photoconversion of FM 1-43 in cultured hippocampal neurons. (A) Phase-contrast image. Region shown by a yellow rectangle was repeatedly imaged in B–E. ✻, Acellular background (see F and G). (B) Fluorescence image after exposure to 15 μM FM 1-43 in 70 mM K⁺ solution for 90 s, followed by washout. Arrowheads, four puncta further analyzed in F and G (not marked). ∗∗, An FM 1-43-negative dendrite (cellular background; see F and G). (C–E) Time course of photoconversion in DAB solution. Elapsed time is shown after start of continuous fluorescence excitation. (F) Absolute bright-field density plotted against time for the 6 ROIs. Upper four lines correspond to ROIs with FM 1-43 fluorescence (arrowheads in B). (G) Data in F transformed into absorbance by using Beer's law (see Materials and Methods). For EM studies, photoconversion reactions were ended within the interval between the broken lines. (Inset) Slopes of change in absorbance. (H) Representative image of a photoconverted bouton, previously subjected to field stimulation (20 Hz for 60 s). Dye exposure during stimulation and an additional 60 s of rest. Arrow points to a PC+ structure that was docked at the active zone. Neurons in A–G and H were 16 and 21 div, respectively.

Figure 2

Density profiles of SVs. (A) Overlay of line density profiles from 8 small clear SVs classified as PC−. No photoconversion was performed. (B) Overlay of line density profiles from 14 small structures classified as PC+. Centers of the lumen (A) and peaks of positive structures (B) were arbitrarily aligned at 40 nm on distance scale. (C) Histogram of luminal density for PC− (open bars) and PC+ SVs (filled bars) from a single photoconverted bouton (Upper) and a negative control (illuminated without prior exposure to FM 1-43, Lower, open bars). (D) Cumulative histograms of luminal density from 5 boutons. Dotted line represents the data from C. Continuous lines represent other boutons taken from the same electron micrograph. (E) Difference in median densities of PC− and PC+ SVs (Δ median density) plotted against proportion of PC+ SVs (%PC+ SVs). Data were obtained from specimens with field stimulation (FS, ●) or with high K⁺ stimulation (○). Linear regression line was drawn for all boutons (r = −0.54, P > 0.1, n = 8). (F) Distributions of SV diameters.

Figure 3

Uniformity of photoconversion within individual boutons, and high variability among different boutons. (A Left) Plots of proportion of PC+ SVs in each section of a bouton. Each symbol represents data from an individual bouton (n = 27). (Right) Filled circles and vertical bars show intrabouton variability, seen in the mean and SD for 3 cases where a bouton was sampled with seven distinct sections (7-section boutons). Filled triangle and vertical bars show interbouton variability, expressed as mean and SD of the mean values for all boutons. (B) Comparison of interbouton variability with different stimulation. Mean and SD are from three specimens. FS, field stimulation (dye exposure during stimulation at 10 Hz for 120 s followed by an additional 60 s of dye exposure, 24 div). High K⁺, high K⁺ application for 90 s (17 and 16 div). Number of boutons examined is indicated.

Figure 4

Correspondence between FM 1-43 fluorescence, photoconversion, and EM. (A) Nomarski differential interference contrast microscopy image of a neuron (43 div). Boxed region was enlarged in B and C. (B) Confocal fluorescence image of boxed region in A. Field stimulation for 40 s at 20 Hz and additional dye exposure for 30 s after stimulus, followed by wash. A rectangle encompasses two fluorescent puncta. (C) Bright-field image of B after photoconversion. (E–G) EM of three thin sections obtained from the region. Numbers 1 and 2 indicate presynaptic structures identified as fluorescent puncta in D. The more fluorescent structure (1) contained more PC+ vesicles. Note that mitochondrion (m) was not photoconverted. Scale bar in C applies to B and C. Scale bar in G applies to E–G.

Figure 5

Analysis of a bouton cluster. (A) Confocal FM 1-43 fluorescence image of neurons (34 div). Field stimulation for 2 s at 10 Hz, followed by 60 s of additional dye exposure. White arrows point to FM 1-43 fluorescent puncta. (B) Corresponding electron micrograph of the same field in A. Mitochondria (m) were photoconverted positively in this sample. ∗ In B denotes a bouton observed in multiple thin sections (1–8). (1–8) Selected serial sections. Arrows in 2 indicate two active zones, visible in panels 1–4. Upper active zone was further seen in 5 and 6. Arrowheads in 4 indicate PC+ SVs. Scale bar under 8 applies to 1–8.