SynapsEM: Computer-Assisted Synapse Morphometry

Abstract

The structural features of a synapse help determine its function. Synapses are extremely small and tightly packed with vesicles and other organelles. Visualizing synaptic structure requires imaging by electron microscopy, and the features in micrographs must be quantified, a process called morphometry. Three parameters are typically assessed from each specimen: (1) the sizes of individual vesicles and organelles; (2) the absolute number and densities of organelles; and (3) distances between organelles and key features at synapses, such as active zone membranes and dense projections. For data to be meaningful, the analysis must be repeated from hundreds to thousands of images from several biological replicates, a daunting task. Here we report a custom computer program to analyze key structural features of synapses: SynapsEM. In short, we developed ImageJ/Fiji macros to record x,y-coordinates of segmented structures. The coordinates are then exported as text files. Independent investigators can reload the images and text files to reexamine the segmentation using ImageJ. The Matlab program then calculates and reports key synaptic parameters from the coordinates. Since the values are calculated from coordinates, rather than measured from each micrograph, other parameters such as locations of docked vesicles relative to the center of an active zone can be extracted in Matlab by additional scripting. Thus, this program can accelerate the morphometry of synapses and promote a more comprehensive analysis of synaptic ultrastructure.

Synaptotagmin 7 is enriched at the plasma membrane through γ-secretase processing to promote vesicle docking and control synaptic plasticity in mouse hippocampal neurons

“…3a-c). In WT synapses, 40% of docked vesicles are lost after a single action potential, as previously reported (Kusick et al, 2020). The number of docked vesicles remains similar 5 ms after HFS, presumably because docked vesicle recovery matches depletion.…”

“…Neurons were grown in Neurobasal-A (Thermofisher; 10888-022) medium supplemented with B-27 (2% Thermofisher; 17504001), Glutamax (2 mM Gibco; 35050061), and pen/strep before experiments. For high-pressure freezing and electron microscopy (EM), cell cultures were prepared on 6-mm sapphire disks (Technotrade), mostly as previously described (Kusick et al, 2020). For two of the three experiments/cultures, genotyping was performed after hippocampal dissection, using cortices, with hippocampi left in NB-A before switching to papain after, while in the third genotyping was performed using tail clips.…”

“…Embedding and sectioning were performed exactly as previously described (Kusick et al, 2020). For ultramicrotomy, 40-nm sections were cut.…”

“…To reconcile these phenotypes, and to gain insight into the underlying mechanisms, we examined SV exocytosis in wild-type and SYT7KO hippocampal synapses in dissociated cultures using an optical biosensor for glutamate (iGluSnFR) (Marvin et al, 2018). Moreover, to gain insights into precise steps in the SV cycle that are regulated by SYT7, we carried out 'zap-and-freeze' (Kusick et al, 2020) electron microscopy experiments. Use of iGluSnFR allowed us to monitor glutamate release directly from single presynaptic nerve terminals, and 'zap-and-freeze' EM yielded novel insights into the membrane trafficking events that occur within 5 ms of an action potential.…”

Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons

“…Only after this randomization were any images excluded from analysis, either because they appeared to not contain a bona fide synapse or the morphology was too poor for reliable annotation. The PM, the active zone, docked SVs, and all SVs in the bouton were annotated in ImageJ using SynapsEM plugins ( Watanabe et al, 2020 ) [ https://github.com/shigekiwatanabe/SynapsEM copy archived at swh:1:rev:11a6227cd5951bf5e077cb9b3220553b506eadbe ( Watanabe, 2021 )]. The active zone was identified as the region of the presynaptic PM with the features described above for identifying a synapse.…”