Tissue multicolor STED nanoscopy of presynaptic proteins in the calyx of Held

Abstract

The calyx of Held, a large glutamatergic terminal in the mammalian auditory brainstem has been extensively employed to study presynaptic structure and function in the central nervous system. Nevertheless, the nanoarchitecture of presynaptic proteins and subcellular components in the calyx terminal and its relation to functional properties of synaptic transmission is only poorly understood. Here, we use stimulated emission depletion (STED) nanoscopy of calyces in thin sections of aldehyde-fixed rat brain tissue to visualize immuno-labeled synaptic proteins including VGluT1, synaptophysin, Rab3A and synapsin with a lateral resolution of approximately 40 nm. Excitation multiplexing of suitable fluorescent dyes deciphered the spatial arrangement of the presynaptic phospho-protein synapsin relative to synaptic vesicles labeled with anti-VGluT1. Both predominantly occupied the same focal volume, yet may exist in exclusive domains containing either VGluT1 or synapsin immunoreactivity. While the latter have been observed with diffraction-limited fluorescence microscopy, STED microscopy for the first time revealed VGluT1-positive domains lacking synapsins. This observation supports the hypothesis that molecularly and structurally distinct synaptic vesicle pools operate in presynaptic nerve terminals.

Figure 1. Schematic representation of the calyx of Held’s architecture.

Diagram illustrating the location of a typical 4 µm brain slice (open bar) with regard to the 3D structure of the calyx of Held (orange) and its postsynaptic cell (blue). A typical cross section through the principal cell and calyx reveals a ring-like arrangement of calyx segments along the circumference of the principal cell (upper right). Active zones (green) are located along the release face of the calyx segments adjacent to the principal cell. The hypothesized localizations of the synaptic proteins investigated in this study are depicted in relation to mitochondria, the presynaptic cytoskeleton and the presynaptic membrane. This ring-like arrangement of presynaptic components illustrates the expected distribution of immunostainings shown in Figures 2 to 7.

Figure 2. Determination of the STED microscope’s resolution.

(A) Image of Nilered beads adsorbed to a glass surface. The center of the green crosshair marks the central point from which twodimensional gauss functions were fit. The inset shows the fitted gaussian distribution of the fluorescence intensities. Scale bar 100 nm, pixel size 8.57 nm. (B) Histogram of the size distributions measured from Nilered beads as described in A. The mean bead size was 33.5 nm, consistent with a diameter of 33 nm theoretically predicted (63×1.3 NA oil, excitation 532 nm, emission 580 nm, STED 647 nm delivers FWHM 30 nm at a 120 mW (80 MHz) STED beam; beads with a physical diameter of 25 nm will then appear to be 33 nm wide). (C) Mowiol-embedded IgG antibodies conjugated to Atto565 adsorbed to a glass surface. 2D-fits were only applied to non-overlapping spots. Scalebar 200 nm. (D) Histogram of the size distributions measured from antibodies using the method described in A. The mean spot size was 43 nm.

Figure 3. Simulation and imaging of synaptic vesicle clusters in the calyx of Held.

(A) Arbitrary cluster of synaptic vescles with a diameter of 50 nm. (B) Illustration of the theoretically expected confocal (B) and STED (C) images of the synaptic vesicle cluster with an assumed resolution of 200 nm and 43 nm, respectively. (D) Profile across the arrows indicated in C reveals FWHM of 63 nm. (E) Raw STED image frame showing individual synaptic vesicles labeled with an anti-VGluT-antibody in a 4 µm thick PFA-fixed brain slice. The average size measured with 2D gauss fits was 61 nm. (F) Confocal image corresponding to E. Scale bars 500 nm.

Figure 4. Nanoscopic synaptophysin distribution in the calyx of Held.

(A) Schematic diagram of synaptophysin localization at synaptic release sites. (B) Confocal and (C) STED images of the synaptophysin distribution within an entire calyx cross section (see Figure 1 for illustration). Wiener filter applied to STED image. (D,E) Magnified views of the regions indicated in B,C. Scale bars 1 µm (B,C) and 500 nm with a pixel size of 19 nm (D,E).

Figure 5. Nanoscopic Rab3A distribution in the calyx of Held.

(A) Schematic diagram of the relative localization of Rab3A at synaptic release sites. (B) Confocal and (C) STED image of Rab3A distribution within a calyx cross-section. Wiener filter applied to STED image. (D,E) Magnified views of the regions indicated in B,C. Scale bars 1 µm. (B,C) and 500 nm with a pixel size of 19 nm (D,E).

Figure 6. Nanoscopic synapsin distribution in the calyx of Held.

(A) Schematic diagram of the presynaptic localization of synapsin. (B,C) Confocal and STED (C,E,G) images of synapsin immunosignal in presynaptic regions of the calyx of Held. Wiener filter applied to STED image (raw data for comparison in Supplemental Figure 1). (D,E,F,G) Magnified views of the regions indicated in B and C. Scale bars 1 µm in B,C and 200 nm in D–G with a pixel size of 19 nm.

Figure 7. Dual color STED nanoscopy of synapsin and VGluT1 distribution in the calyx of Held.

(A) Schematic diagram depicting the hypothesized interplay of synapsin and synaptic vesicles (VGluT1) in a glutamatergic terminal. (B) Dual-color confocal and (C) STED images of synapsin and VGluT1 distribution in the calyx. Scale bar 1 µm. (D,E,F) Magnified views of the subregions marked in C in clockwise order of their appearance. Scale bar 500 nm with a pixel size of 19 nm.