Synaptic and Somatic Targeting of ArcLight, a Genetically Encoded Voltage Indicator

ABSTRACT Voltage signals in neurons are highly compartmentalized, which can influence their specific functions within neuronal circuits. Targeting of a genetically encoded voltage indicator (GEVI) to specific subcellular compartments can enhance the signal-to-noise ratio and provide more precise information about the location and timing of synaptic firing across different neuronal regions, reducing spatiotemporal signal convolution. To achieve subcellular targeting of the GEVI, ArcLight, we utilized five different postsynaptic targeting sequences (Shaker K+ channel C-terminus, stargazin C-terminus, rat Neuroligin-1 C-terminus, and anti-homer1 nanobodies HC20 & HC87) to direct ArcLight expression to the excitatory postsynaptic density. Additionally, we assessed a presynaptic-targeting tag (rat Neurexin-1β C-terminus) and a somatodendritic targeting tag (Kv2.1-Lk-Tlcn C-terminus). Patch clamp experiments in HEK293 cells showed that the targeting tags used in this study did not significantly alter ArcLight’s voltage sensitivity compared to controls. AAV infection in the mouse olfactory bulb demonstrated that the subcellular targeting sequences effectively localized GEVI expression to specific compartments of mitral/tufted cells, including postsynaptic densities, presynaptic terminals, and somatodendritic regions. Furthermore, in vivo voltage imaging in mice expressing targeting-enhanced ArcLight variants revealed odorant-evoked responses similar to those observed with the original ArcLight. This indicates that subcellular targeting did not significantly impact the voltage sensing capability of ArcLight in mitral/tufted cells. Competing Interest Statement The authors have declared no competing interest. Abbreviations - PSD - postsynaptic density - ONL - Olfactory Neuron Layer - GL - Glomerular Layer - EPL - External Plexiform Layer - dEPL - dorsal EPL - vEPL - ventral EPL - MCL - Mitral Cell Layer - IPL - inner Plexiform Layer - GCL - Granule Cell Layer - rNxn - rat Neurexin-1β C-terminal tag - KvLkTn - Kv2.1-linker-Tlcn C-terminal tag - SKC - Shaker K+ channel C-terminal tag - rNL1 - rat Neuroligin-1 C-terminal tag - Stgz - stargazin C-terminal with phosphomimetic charge mutations - HC20 - HC20 anti-homer1 nanobody tag - HC87 - HC87 anti-homer1 nanobody tag.