Abstract
While electron microscopy represents the gold standard for detection of synapses, a number of limitations prevent its broad applicability. 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 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. Here, we delineate a macro that uses open-source and freely available ImageJ or FIJI for analysis of multichannel, z-stack confocal images. 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. Puncta from each channel are used to eliminate non-apposed puncta and are subsequently linked with their cognate from the other channel. 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). Using gephyrin as an inhibitory, post-synaptic scaffolding protein, we identify inhibitory synapses in basolateral amygdala, central amygdala, arcuate and the ventromedial hypothalamus. Systematic variation of the settings identify the parameters most critical for this analysis. Identification of specifically overlapping puncta allows for correlation of morphometry data between each channel. Finally, we extend the analysis to only examine puncta overlapping with a cytoplasmic marker of specific cell types, a distinct advantage beyond electron microscopy. Bassoon puncta are restricted to virally transduced, pedunculopontine tegmental nucleus (PPN) axons expressing yellow fluorescent protein. NR1 puncta are restricted to tyrosine hydroxylase labeled dopaminergic neurons of the substantia nigra pars compacta (SNc). The macro identifies bassoon-NR1 overlap throughout the image, or those only restricted to the PPN-SNc connections. Thus, we have extended the available analysis tools that can be used to study synapses in situ. Our analysis code is freely available and open-source allowing for further innovation.
Highlights
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)
The macro first identifies the presynaptic puncta from one color channel and the post-synaptic puncta from a second color channel from the same image (Figure 1A)
We found a negative correlation between threshold and puncta density, with far more puncta identified with lower thresholds and fewer puncta identified at higher thresholds (Figure 2E)
Summary
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). 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. To identify these two components using fluorescent microscopy requires the use of antibodies that selectively identify a protein from each compartment
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