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.
Highlights
Understanding the mechanisms of synaptic transmission requires detailed characterizations of synapses at the ultrastructural level
Ultrastructural analysis of synapses has been performed by many labs over the years
For example, IMOD (Kremer et al, 1996), TrakEM2 (Cardona et al, 2012), and Reconstruct (Fiala, 2005; SynapseWeb, Kristen Harris), provide visualization software for data acquired from either serial sections or tomograms
Summary
Understanding the mechanisms of synaptic transmission requires detailed characterizations of synapses at the ultrastructural level. A subset of vesicles are docked, that is, in contact with the active zone membrane by morphology (Schikorski and Stevens, 1997; Hammarlund et al, 2007; Imig et al, 2014), and fuse in response to calcium influx (Heuser et al, 1979; Heuser and Reese, 1981) Following fusion, these vesicles are recycled locally via endocytosis and components sorted in an endosome to sustain synaptic transmission (Ceccarelli et al, 1972; Heuser and Reese, 1973; Dittman and Ryan, 2009; Saheki and De Camilli, 2012; Watanabe et al, 2013a,b, 2014; Kononenko and Haucke, 2015). Synaptic morphometry requires the resolution of electron microscopy
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