Abstract
Mapping and determining the molecular identity of individual synapses is a crucial step towards the comprehensive reconstruction of neuronal circuits. Throughout the history of neuroscience, microscopy has been a key technology for mapping brain circuits. However, subdiffraction size and high density of synapses in brain tissue make this process extremely challenging. Electron microscopy (EM), with its nanoscale resolution, offers one approach to this challenge yet comes with many practical limitations, and to date has only been used in very small samples such as C. elegans, tadpole larvae, fruit fly brain, or very small pieces of mammalian brain tissue. Moreover, EM datasets require tedious data tracing. Light microscopy in combination with tissue expansion via physical magnification—known as expansion microscopy (ExM)—offers an alternative approach to this problem. ExM enables nanoscale imaging of large biological samples, which in combination with multicolor neuronal and synaptic labeling offers the unprecedented capability to trace and map entire neuronal circuits in fully automated mode. Recent advances in new methods for synaptic staining as well as new types of optical molecular probes with superior stability, specificity, and brightness provide new modalities for studying brain circuits. Here we review advanced methods and molecular probes for fluorescence staining of the synapses in the brain that are compatible with currently available expansion microscopy techniques. In particular, we will describe genetically encoded probes for synaptic labeling in mice, zebrafish, Drosophila fruit flies, and C. elegans, which enable the visualization of post-synaptic scaffolds and receptors, presynaptic terminals and vesicles, and even a snapshot of the synaptic activity itself. We will address current methods for applying these probes in ExM experiments, as well as appropriate vectors for the delivery of these molecular constructs. In addition, we offer experimental considerations and limitations for using each of these tools as well as our perspective on emerging tools.
Highlights
Historical Perspective of ConnectomicsEmbedded in the concept of the ‘‘neuron doctrine’’ is the principle that neurons communicate through synapses, a striking assumption first made by Ramon y Cajal over a century ago (Ramón y Cajal, 1909)
We summarize some major molecular strategies for visualizing the synapse at high resolution that can be used in combination with expansion microscopy (ExM) for optical connectome
The multiplexing approach can significantly increase the utility of ExM for molecular profiling of synapses, it still needs to be carefully validated for a large variety of synaptic proteins
Summary
Embedded in the concept of the ‘‘neuron doctrine’’ is the principle that neurons communicate through synapses, a striking assumption first made by Ramon y Cajal over a century ago (Ramón y Cajal, 1909). The very recent modification of MAP, denoted epitopepreserving MAP or eMAP, was optimized to achieve maximal preservation of antigenicity in mouse and marmoset brain tissue, increasing success rates of staining with synaptic antibodies to more than 94% (Park et al, 2021) Another advantage of post-expansion immunostaining is the ability to carry out multiple rounds of labeling and imaging, providing an unprecedent degree of multiplexed superresolution synaptic proteomic profiling. In addition to identifying the synapse itself, an important part of connectomics is tracing synaptic connections to the originating neuron From this perspective, the expression of fluorescent proteins might be a good alternative to antibodies, as the visualization of genetically encoded fluorescent probes does not require an additional staining step and they can be evenly expressed throughout the plasma membrane and/or cytoplasm. The expression of fluorescent proteins using the rabies virus is a powerful transneuronal tracing technology (Ugolini, 2011; Kim et al, 2016)
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