Abstract
We combine a chemically-synthesized, voltage-sensitive fluorophore with a genetically encoded, self-labeling enzyme to enable voltage imaging in Drosophila melanogaster. Previously, we showed that a rhodamine voltage reporter (RhoVR) combined with the HaloTag self-labeling enzyme could be used to monitor membrane potential changes from mammalian neurons in culture and brain slice. Here, we apply this hybrid RhoVR-Halo approach in vivo to achieve selective neuron labeling in intact fly brains. We generate a Drosophila UAS-HaloTag reporter line in which the HaloTag enzyme is expressed on the surface of cells. We validate the voltage sensitivity of this new construct in cell culture before driving expression of HaloTag in specific brain neurons in flies. We show that selective labeling of synapses, cells, and brain regions can be achieved with RhoVR-Halo in either larval neuromuscular junction (NMJ) or in whole adult brains. Finally, we validate the voltage sensitivity of RhoVR-Halo in fly tissue via dual-electrode/imaging at the NMJ, show the efficacy of this approach for measuring synaptic excitatory post-synaptic potentials (EPSPs) in muscle cells, and perform voltage imaging of carbachol-evoked depolarization and osmolarity-evoked hyperpolarization in projection neurons and in interoceptive subesophageal zone neurons in fly brain explants following in vivo labeling. We envision the turn-on response to depolarizations, fast response kinetics, and two-photon compatibility of chemical indicators, coupled with the cellular and synaptic specificity of genetically-encoded enzymes, will make RhoVR-Halo a powerful complement to neurobiological imaging in Drosophila.
Highlights
Voltage imaging in intact brains offers the tantalizing promise to watch, in real time, the electrical changes that underlie physiology
We recently reported a chemical-genetic hybrid, in which a chemically-synthesized rhodamine-based voltage reporter (RhoVR) (Deal et al, 2016) attached to a flexible polyethyleneglycol (PEG) linker terminating with a chloroalkane forms a covalent bond with a cell-expressed HaloTag (Figure 1), enabling voltage imaging from defined neurons, in mouse cortical brain slices (Deal et al, 2020)
For the first time, that RhoVR-Halo dyes can label specific neurons in vivo and that voltage changes can be visualized using epifluorescence microscopy at synapses in the neuromuscular junction (NMJ) and whole-brain explants
Summary
Voltage imaging in intact brains offers the tantalizing promise to watch, in real time, the electrical changes that underlie physiology. We recently reported a chemical-genetic hybrid, in which a chemically-synthesized rhodamine-based voltage reporter (RhoVR) (Deal et al, 2016) attached to a flexible polyethyleneglycol (PEG) linker terminating with a chloroalkane forms a covalent bond with a cell-expressed HaloTag (Figure 1), enabling voltage imaging from defined neurons, in mouse cortical brain slices (Deal et al, 2020) This approach, RhoVRHalo, takes advantage of the fast kinetics, linear turn-on response, and 2P compatibility of RhoVR-type indicators (Kulkarni et al, 2018; Kazemipour et al, 2019), and pairs it with the ability to target specific cells using traditional genetic methods. When paired with voltage-sensitive RhoVR-Halo (Deal et al, 2020), HaloTag-expressing flies allow cell type-specific labeling in vivo, and voltage imaging in a variety of contexts, including synaptic imaging at the neuromuscular junction (NMJ) and across defined neuronal populations in fly brain explants
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