![]() ![]() ![]() Rather, the presence of an active zone release site, seen as an electron dense area of the pre-synaptic plasma membrane in an electron microscopy micrograph, is what defines a synapse ( Beaudoin et al., 2012 Burette et al., 2015). ![]() Additionally, synaptic vesicles alone do not demonstrate a synapse using electron microscopy. However, synaptic vesicles are known to be in parts of the axon away from the release site ( Staras and Branco, 2010). Synapses were originally identified by proteins found on synaptic vesicles, a necessary component of a synapse ( Bixby and Reichardt, 1985). The identification of a protein that selectively and specifically targets a pre- or post-synaptic structure requires careful consideration. To identify these two components using fluorescent microscopy requires the use of antibodies that selectively identify a protein from each compartment. The unique challenge of synaptic identification is that a synapse is defined as the zone of interaction between two neurons, where one neuron releases neurotransmitter and the other neuron receives the neurotransmitter. The standard has been to rely on electron microscopy for accurate identification of synapses, and this analysis was done visually by a user, though this is increasingly being done by computers ( Merchan-Perez et al., 2009 Beaudoin et al., 2012 Kornfeld and Denk, 2018 Nosov et al., 2020 Pourhoseini et al., 2021). However, identification of chemical synapses, henceforth referenced to simply as a synapse, from imaging data represents a unique challenge. Many kinds of information lend themselves to simple means of quantification, including dendritic complexity being analyzed by Sholl analysis, protein expression level by intensity analysis, or co-localization of two proteins by correlation analysis ( Beaudoin et al., 2012 Nelson et al., 2016 Boulan et al., 2020 Ligon et al., 2020). Quantification of digitally acquired images is a field standard for any microscopic data (For example: Beaudoin et al., 2012 Taylor et al., 2020 Pourhoseini et al., 2021). Our analysis code is freely available and open-source allowing for further innovation. Thus, we have extended the available analysis tools that can be used to study synapses in situ. The macro identifies bassoon-NR1 overlap throughout the image, or those only restricted to the PPN-SNc connections. NR1 puncta are restricted to tyrosine hydroxylase labeled dopaminergic neurons of the substantia nigra pars compacta (SNc). Bassoon puncta are restricted to virally transduced, pedunculopontine tegmental nucleus (PPN) axons expressing yellow fluorescent protein. Finally, we extend the analysis to only examine puncta overlapping with a cytoplasmic marker of specific cell types, a distinct advantage beyond electron microscopy. Identification of specifically overlapping puncta allows for correlation of morphometry data between each channel. Systematic variation of the settings identify the parameters most critical for this analysis. Using gephyrin as an inhibitory, post-synaptic scaffolding protein, we identify inhibitory synapses in basolateral amygdala, central amygdala, arcuate and the ventromedial hypothalamus. These methods are applied to analysis of a pre-synaptic marker, bassoon, with two different post-synaptic markers, gephyrin and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1). Puncta from each channel are used to eliminate non-apposed puncta and are subsequently linked with their cognate from the other channel. With use of a high magnification with a high NA objective, we outline two methods to identify puncta in either sparsely or densely labeled images. Here, we delineate a macro that uses open-source and freely available ImageJ or FIJI for analysis of multichannel, z-stack confocal images. While immunostaining and imaging techniques have improved to allow for identification of synapses in tissue, analysis and identification of these appositions are not facile, and there has been a lack of tools to accurately identify these appositions. A key method for detecting synapses is immunostaining for markers of pre- and post-synaptic proteins, which can infer a synapse based upon the apposition of the two markers. While electron microscopy represents the gold standard for detection of synapses, a number of limitations prevent its broad applicability. Department of Biology, Trinity University, San Antonio, TX, United States. ![]()
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