SynLight: a bicistronic strategy for simultaneous active zone and cell labeling in the Drosophila nervous system

• Published in: G3 : genes - genomes - genetics Vol. 13; no. 11
• Main Authors: Aimino, Michael A, Humenik, Jesse, Parisi, Michael J, Duhart, Juan Carlos, Mosca, Timothy J
• Format: Journal Article
• Language: English
• Published: US Oxford University Press 01.11.2023
• Subjects: Animals; Drosophila - metabolism; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Motor Neurons - metabolism; Neurogenetics; Neuromuscular Junction - metabolism; Peptides; Synapses - genetics; 2A peptides; synapse; neuromuscular junction; fluorescent labeling; optic lobe; antennal lobe; Drosophila
• ISSN: 2160-1836; 2160-1836
• DOI: 10.1093/g3journal/jkad221

Abstract At synapses, chemical neurotransmission mediates the exchange of information between neurons, leading to complex movement, behaviors, and stimulus processing. The immense number and variety of neurons within the nervous system make discerning individual neuron populations difficult, necessitating the development of advanced neuronal labeling techniques. In Drosophila, Bruchpilot-Short and mCD8-GFP, which label presynaptic active zones and neuronal membranes, respectively, have been widely used to study synapse development and organization. This labeling is often achieved via the expression of 2 independent constructs by a single binary expression system, but expression can weaken when multiple transgenes are expressed by a single driver. Recent work has sought to circumvent these drawbacks by developing methods that encode multiple proteins from a single transcript. Self-cleaving peptides, specifically 2A peptides, have emerged as effective sequences for accomplishing this task. We leveraged 2A ribosomal skipping peptides to engineer a construct that produces both Bruchpilot-Short-mStraw and mCD8-GFP from the same mRNA, which we named SynLight. Using SynLight, we visualized the putative synaptic active zones and membranes of multiple classes of olfactory, visual, and motor neurons and observed the correct separation of signal, confirming that both proteins are being generated separately. Furthermore, we demonstrate proof of principle by quantifying synaptic puncta number and neurite volume in olfactory neurons and finding no difference between the synapse densities of neurons expressing SynLight or neurons expressing both transgenes separately. At the neuromuscular junction, we determined that the synaptic puncta number labeled by SynLight was comparable to the endogenous puncta labeled by antibody staining. Overall, SynLight is a versatile tool for examining synapse density in any nervous system region of interest and allows new questions to be answered about synaptic development and organization. Having access to more efficient tools for visualizing synapses will enable more nuanced research on synaptic development or the causes of synapse dysfunction. SynLight labels both the membranes of neurons as well as their synapses from a single open reading frame with single neuron population specificity, allowing future experimental designs to include additional effectors and markers.