Advanced Fluorescence Protein-Based Synapse-Detectors

Abstract

The complex information-processing capabilities of the central nervous system emerge from intricate patterns of synaptic input-output relationships among various neuronal circuit components. Understanding these capabilities thus requires a precise description of the individual synapses that comprise neural networks. Recent advances in fluorescent protein engineering, along with developments in light-favoring tissue clearing and optical imaging techniques, have rendered light microscopy (LM) a potent candidate for large-scale analyses of synapses, their properties, and their connectivity. Optically imaging newly engineered fluorescent proteins (FPs) tagged to synaptic proteins or microstructures enables the efficient, fine-resolution illumination of synaptic anatomy and function in large neural circuits. Here we review the latest progress in fluorescent protein-based molecular tools for imaging individual synapses and synaptic connectivity. We also identify associated technologies in gene delivery, tissue processing, and computational image analysis that will play a crucial role in bridging the gap between synapse- and system-level neuroscience.

Keywords: fluorescent protein sensors; gene delivery; light microscopy; mapping and localization; synapses; synaptic connectivity.

Figure 1

Scheme of single component synapse detectors. (A) Graphical depiction of the conventional green fluorescent protein (GFP) tagging scheme and summary of the expressed carrier-sensor construct. GFP (green circle) and other fluorescent proteins (FPs) are directly tagged to synaptic proteins in the presynaptic terminal or postsynaptic spines. Major targets for tagging include presynaptic vesicle proteins (e.g., vesicle-associated membrane protein 2 (VAMP2) and synaptophysin), postsynaptic receptors (e.g., α-amino-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, AMPAR) and postsynaptic density protein-95 (PSD-95). (B) pH-sensitive FP mutants are fused to synaptic vesicle membrane proteins, such as VAMP2, to visualize vesicle secretion/recycling and neurotransmission. A pH-sensitive GFP variant, pHluorin does not fluoresce (gray circle) when inside the acidic chemical environment of the synaptic vesicle, but becomes highly fluorescent (green circle) when the vesicle is released and exposed to the neutral extracellular environment. (C) Inhibition of Synaptic Release with CALI (InSynC): attached to target SNARE proteins, molecular actuators such as mini small singlet oxygen generator (miniSOG; light blue filled-in circle) selectively inactivate specific synaptic proteins that regulate vesicle release and other synaptic events. When illuminated with blue light, miniSOG stimulates generation of reactive oxygen species (small, dark blue filled-in circles), which then oxidizes susceptible amino acid residues in target vesicle proteins and deactivates protein functions. (D) TimeSTAMP effectively tracks spatiotemporally controlled protein synthesis and trafficking in living neurons. In the presence of a membrane-permeable protease inhibitor, NS3 protease (gray oval) activity is inhibited, and Venus C-terminus (Venus CT) and Venus N-terminus (Venus NT) reconstitute as fluorescent Venus (yellow circle). Reconstituted Venus accumulates in the postsynaptic spine, the trafficking destination of the fused PSD-95. When the protease inhibitor is present, however, NS3 protease cleaves the protease target sites (gray circles flanking NS3), preventing Venus reconstitution.

Figure 2

Scheme of dual component synapse detectors. (A) GFP reconstitution-dependent molecular tools detect pre- and postsynaptic membrane apposition through the reconstitution of spGFP fragments each attached to the extracellular ends of membrane protein carriers. Synapse-targeted GFP Reconstitution Across Synaptic Partners (GRASP) utilizes a presynaptic component of spGFP11 (spG11, circular wedge) linked to a CD4-neurexin-1β C-terminus (NxCT) fusion through flexible peptide linkers (gray zigzag), and a postsynaptic component of spGFP1-10 (spG1-10) and truncated neuroligin-1 (tNg) connected with a linker. SpG11 and spG1-10 unite at synapses, not random membrane contacts. In activity-dependent X-RASP the presynaptic split-XFP1-10 (spX1-10) is fused to SNARE proteins such as synaptobrevin (syb), and unites with CD4-bound spG11 when presynaptic activity triggers vesicle release. (B) Neurexin (Nx)-neuroligin (Ng) interaction-dependent molecular tools detect individual synapses through fluorescent protein (FP)-based (SynView) and enzymatic reaction-based (Interaction-Dependent Probe Incorporation Mediated by Enzymes, ID-PRIME) direct visualization of Nx-Ng interaction. In SynView, spG11 is inserted between the esterase and proximal extracellular domain of Ng, and spG1-10 in the middle of the LNS domain of Nx. GFP reconstitutes when Nx and Ng unites. ID-PRIME uses the identical set of synaptic protein carriers, but tandem LAP tags (3xLAP) and lipoic acid ligase A (LplA) are attached to C-terminus of Ng esterase domain and Nx LNS domain, respectively. Nx-Ng interaction initiates LplA-mediated lipoic acid tagging to 3xLAP, and signals useful for imaging originate from antibody-fluorophore conjugates.

Figure 3

Convergence of technologies for synaptic neuroscience at the systems level. (A) Synapse-detector genes can be delivered to target neural components through germline manipulation-based and viral system-based methods. Exogenous genetic materials can be effectively introduced to the germline with high expression specificity via Cre-mediated conditional knock-in (KI) strategies and the homology directed repair (HDR) pathway of the clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas system. In a Cre-mediated conditional KI strategy called endogenous labeling via exon duplication (ENABLED), a KI cassette including duplicates of exons 19 and 20 and the 3’UTR of PSD-95 is generated. The first duplicate is flanked by head-to-tail oriented loxP sites (black arrows), while the second duplicate has monovalent Venus (mV) inserted between exon 20 and the 3’UTR. The two duplicate sequences, along with exon 18, are flanked by a set of homology arms (h arm), which mediates KI cassette insertion through homologous recombination. Cre-lox recombination excises the first duplicate containing translation stop signals and polyadenylation sequences, and activates mV expression only in Cre-expressing neurons. Transient expression of sensor genes using viral vectors benefits from efforts to engineer expression through Cre-dependent expression cassettes e.g., Cre-dependent Mammalian GRASP (mGRASP) constructs and newly engineered serotypes that have more selective tropism and transduction efficiency (e.g., AAV-PHP.B). Local tissue or systemic injection of such viral systems can lead to flexible, versatile gene delivery in mature organisms. (B) Combination of light-favoring brain clearing, whole-brain imaging, and computational techniques for three-dimensional synapse mapping enables single-synapse level analysis of synaptic profiles across the whole brain. Further improvements in lipid extraction, refractive index matching, advanced light-sheet microscopy, and large-scale data processing and 3D reference space generation will accelerate systems neuroscience at the synaptic scale.