Visualizing Synapses

The use of fluorescent proteins (such as Green Fluorescent Protein (GFP) and its derivatives) has revolutionized the study of the cellular biology of synaptogenesis. Fusion proteins between synaptic components and GFP allow in vivo analysis of the synapse in translucent fly larvae and worm. These fusion proteins have also served as powerful screening tools for mutants that affect the distribution of synaptic components.

A key reagent used to examine C. elegans NMJs is the Synaptobrevin::GFP fusion reporter5 (Figure 3.1C). Synaptobrevin (SNB-1) is an integral membrane protein of SVs. SV pools are visualized by SNB::GFP, whose pattern correlates with that of most synapses. A SYD-2::GFP (SYnapse Defective::GFP) fusion reporter is also used to visualize active zones in multiple types of NMJs7.

SYD-2 is a Liprin- a protein important for active zone assembly (see Section 5.2.3)6. Several fluorescently tagged presynaptic proteins have also recently been used to visualize synaptic contacts8.

4.1.2. Drosophila

In contrast to live imaging approaches used to visualize the developing synapse in C. elegans, visualization of Drosophila NMJs has traditionally relied on a bank of monoclonal antibodies9. For example, anti-HRP is commonly used to label neurons (Figure 3.1D). However, several GFP reagents have also been developed for live observations. CD8::GFP::Sh is used to visualize the postsynaptic muscle of boutons. This protein contains the extracellular and transmembrane domains of the human T lymphocyte protein (CD8), GFP, and the cytoplasmic C-terminal sequence of the Shaker potassium channel (Sh). The CD8::Sh protein is expressed specifically at the postsynaptic muscle10. In addition, proteins such as synaptophysin::GFP and synaptotagmin::GFP (SV-associated proteins) as well as SNB::GFP are commonly used to visualize presynaptic terminals. These fluorescent molecules have enabled characterization of target recognition and synapse formation.

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