High-Pressure Freezing EM Tomography of Entire Ribbon Synapses in the Retina

In the retina, presynaptic active zones in photoreceptors and bipolar cells are distinguished by a plate-like "ribbon" linked to the plasma membrane (PM) and surrounded by dozens of synaptic vesicles (SVs) tethered to it. SVs at the base of the ribbon, closest to the PM, are thought to constitute the readily releasable vesicle pool (RRP), i.e., SVs primed to be released 1-2 ms following stimulation. The number of SVs in the RRP is a critical synaptic parameter that influences synaptic strength and varies with light levels to enable ribbon synapses to compute visual information. Physiological RRP measurements agree well with anatomical estimates obtained via electron microscopy (EM), although EM often employs chemical fixation, which causes exocytotic artifacts that may influence RRP size. Here, we used EM tomography to compare anatomical features of rod bipolar cell ribbon synapses in retinas, from rats of either sex, prepared by chemical fixation or high-pressure freezing (HPF), a technique that minimizes artifactual exocytosis. We found that ribbon dimensions and the number of ribbon-associated SVs were roughly similar in the two preparations. Cleft volume and SV diameter were larger in HPF-prepared tissue, however, and vesicles at the base of the ribbon were more closely associated with the PM, suggesting morphologically distinguishable steps in the SV priming process. The distribution of SV diameters was positively skewed in both conditions, but larger SVs were too scarce to explain previous electrophysiological results, which likely reflect multivesicular release at the PM rather than prefusion of SVs prior to exocytosis.