Abstract
Synaptic transmission has been studied for decades, as a fundamental step in brain function. The structure of the synapse, and its changes during activity, turned out to be key aspects not only in the transfer of information between neurons, but also in cognitive processes such as learning and memory. The overall synaptic morphology has traditionally been studied by electron microscopy, which enables the visualization of synaptic structure in great detail. The changes in the organization of easily identified structures, such as the presynaptic active zone, or the postsynaptic density, are optimally studied via electron microscopy. However, few reliable methods are available for labeling individual organelles or protein complexes in electron microscopy. For such targets one typically relies either on combination of electron and fluorescence microscopy, or on super-resolution fluorescence microscopy. This review focuses on approaches and techniques used to specifically reveal synaptic organelles and protein complexes, such as cytoskeletal assemblies. We place the strongest emphasis on methods detecting the targets of interest by affinity binding, and we discuss the advantages and limitations of each method.
Highlights
Chemical synapses support neurotransmission by releasing neurotransmitter from the presynaptic side, and responding to it on the postsynaptic side
This approach is typical of concepts such as photo-activated localization microscopy (PALM; Betzig et al, 2006), stochastic optical reconstruction microscopy (STORM and dSTORM; Rust et al, 2006; van de Linde et al, 2011), or ground state depletion microscopy followed by individual molecule return (GSDIM; Testa et al, 2010)
green fluorescent protein (GFP)-labeled actin-binding domains have a number of advantages over direct actin-GFP fusions or GFP-nanobodies: they predominantly bind to actin filaments and not actin monomers, allowing to visualize the filaments with less background; do not impair actin polymerization as toxins targeting actin do, better preserving native cytoskeleton architecture and allowing to study its dynamics; and do not change the total concentration of actin monomers in the cell, what could otherwise affect cellular physiology through sequestering of actin-binding proteins, initiation of polymerization or other mechanisms (Figure 5F)
Summary
Chemical synapses support neurotransmission by releasing neurotransmitter from the presynaptic side, and responding to it on the postsynaptic side. A widely used approach to visualize cellular components is to use genetically encoded fluorescent tags [such as green fluorescent protein (GFP) and its derivatives] fused to proteins of interest with consequent imaging with fluorescent microscopy This requires protein overexpression or genome editing but results in a high labeling density, and enables live cell imaging of tagged molecules. This has raised substantially the interest in fluorescent probes that bind to molecules of interest, and that can be conjugated to specific chemical dyes Such elements are commonly used to visualize endogenous cellular components at their native locations, and enable investigators to exploit recent advances in super-resolution microscopy, thereby combining the two main advantages of the methods described above: labeling of cellular components with high specificity and efficiency, and nanometer resolution (Hell and Wichmann, 1994; Hofmann et al, 2005; Rust et al, 2006; Sharonov and Hochstrasser, 2006). We will discuss the most prominent ones, their mechanisms of action, main advantages, and disadvantages
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