Although HaloTag and other self-labeling enzymes have been successfully expressed in transgenic flies, the reported lines localize HaloTag intracelluarly (Kohl et al., 2014; Sutcliffe et al., 2017; Meissner et al., 2018; Abdelfattah et al., 2019). Our first task was to generate a HaloTag that expressed on the extracellular face of membranes. Previous chemical-genetic hybrids deployed in mammalian cells used a transmembrane domain from the platelet-derived growth factor receptor (PDGFR) to localize HaloTag to the cell surface and a secretion signal peptide from immunoglobulin K (IgK) to enhance export of the construct (Deal et al., 2020). To adapt HaloTag-directed chemical-genetic hybrids for voltage imaging in Drosophila, we selected CD4 as a transmembrane anchor, on account of its good membrane association in Drosophila neurons (Han et al., 2011), fusing it to the C-terminus of the HaloTag. We sub-cloned this construct into different vectors for expression in mammalian (pcDNA3.1) and insect cells (pJFRC7) (Pfeiffer et al., 2010).

HaloTag-CD4 shows good expression on cell surfaces. In mammalian cells, CD4 localizes to the cell surface by anti-CD4 immunocytochemistry (Supplementary Figure 1). Inclusion of the self-labeling enzyme, HaloTag, affords the opportunity to confirm not only localization, but activity of the expressed enzyme by delivering HaloTag substrates. HEK cells expressing HaloTag-CD4 and treated with RhoVR-Halo (100 nM) show good membrane localization (Figure 2a), while cells that do not express HaloTag-CD4 show approximately 3.5-fold lower fluorescence levels (Figures 2b,c). RhoVR-Halo survives fixation: following live-cell imaging, cells can be fixed and retain their RhoVR-Halo staining, which serves as a useful counterstain to the anti-CD4 immunocytochemistry (Supplementary Figure 1). Live cells labeled with RhoVR-Halo and subsequently fixed, permeabilized with detergent and assayed for CD4 via immunochemistry reveal the majority of CD4 is found intracellularly. Confocal imaging shows that RhoVR-Halo localizes to cell membranes (Supplementary Figures 2A–C). HaloTag-mediated labeling works with other dyes, too: HEK cells expressing HaloTag-CD4 and labeled with TMR-Halo show approximately 15-fold greater fluorescence than cells that do not express HaloTag-CD4 (Supplementary Figure 2).

In S2 cells, an immortalized Drosophila cell line, we also observe cell surface localization of HaloTag-CD4, as visualized by anti-CD4 immunocytochemistry (Supplementary Figure 3). S2 cells show similar HaloTag-CD4 dependent staining with TMR-Halo (100 nM, Figure 3 and Supplementary Figures 3A,B) with a 20-fold enhancement in fluorescence intensity in HaloTag-CD4 expressing cells compared to non-expressing cells (Figure 3c). TMR-Halo staining in S2 cells is also retained post-fixation (Supplementary Figure 3).

We evaluated the voltage sensitivity of RhoVR-Halo in HaloTag-CD4 expressing HEK293T cells (Figure 4). After loading cells with RhoVR-Halo (500 nM), cells were subjected to whole-cell, patch-clamp electrophysiology. The voltage sensitivity of RhoVR-Halo in HaloTag-CD4 expressing HEK293T cells is approximately 14% per 100 mV (± 2%, SEM n = 7 cells). This is approximately 70% of the value we obtained when HaloTag is targeted with previously developed (Deal et al., 2020) HaloTag-pDisplay (Supplementary Figure 4).

To evaluate the performance of cell surface-expressed HaloTag-CD4 in intact flies, we generated transgenic flies (BestGene Inc.) and crossed the resulting UAS-HaloTag-CD4 line with a pan-neuronal driver line, neuronal synaptobrevin-GAL4 (nSyb-GAL4) (DiAntonio et al., 1993), which was used to drive HaloTag-CD4 expression in all neurons, (Figure 5a). Brains of nSyb-GAL4 > HaloTag-CD4 flies show strong CD4 expression (Figures 5a–c). The pattern of anti-CD4 fluorescence indicates good localization to the plasma membrane (Figures 5d,e).

To evaluate labeling specificity, we expressed UAS-HaloTag-CD4 in a subset of neurons. We crossed UAS-HaloTag-CD4 flies with GH146-GAL4 flies (Stocker et al., 1997; Wilson et al., 2004) to drive expression in a subpopulation of olfactory projection neurons (PNs) in the antennal lobe, a key olfactory relay. Immunohistochemistry reveals strong CD4 staining, localized to the antennal lobe in transgenic GH146-GAL4 > HaloTag-CD4 flies (Figures 5f–h). These neurons also showed good extracellular staining (Figures 5i–l).

HaloTag remains functional when expressed on the cell surface of Drosophila neurons, enabling a range of brain regions and neurons to be labeled with small molecules. We delivered TMR-Halo (1 μM) to live flies via application of a solution of TMR-Halo in artificial hemolymph (AHL) to flies with their cuticle removed (Harris et al., 2015) (see Supplementary Material for dissection details). We then imaged via confocal microscopy to establish the extent of labeling (Figure 6). In GH146-GAL4 > HaloTag flies (PN labeling) treated with TMR-Halo, we observe strong fluorescence localized to the antennal lobe (Figure 6a). Non-transgenic fly controls show low fluorescence levels in the brain and antennal lobe (GH146-GAL alone, Figure 6b). TMR-Halo in combination with HaloTag-CD4 can be used to label single cells. VT011155-GAL4 > HaloTag-CD4 fly brains drive expression in single interoceptive subesophageal zone neurons (ISNs) (Jourjine et al., 2016), and treatment with TMR-Halo results in bright fluorescence localized to these neurons (Figure 6c). Similar staining profiles can be achieved with the voltage-sensitive RhoVR-Halo, which clearly labels PNs of the antennal lobe (Figure 6d, GH146-GAL4). High magnification examination of labeled projection neurons reveals membrane-localized staining (Figures 6e,g, red) alongside Hoechst 33342 nuclear staining (Figures 6f,h, blue). RhoVR-Halo can also label smaller sub-sets of neurons cells; treatment of Nan-GAL4 > HaloTag-CD4 brains with RhoVR-Halo results in labeling of ISNs (Figure 6i).

We used the same live-animal staining procedure to optimize the loading of RhoVR-Halo (Supplementary Figure 5). We find that 2 μM RhoVR-Halo provides good staining in the antenna lobes of GH146-GAL4 > HaloTag-CD4 crosses (Supplementary Figure 5). Fluorescence from RhoVR-Halo is localized to the periphery of cell bodies, again supporting the extracellular expression of HaloTag-CD4 (Figures 6e–h). Compared to regions of the brain that do not express HaloTag-CD4, RhoVR-Halo fluorescence is approximately three times higher (Supplementary Figures 5B,C). We find homozygous flies for GH146-GAL4 > HaloTag-CD4 have slightly higher fluorescence levels compared to levels of heterozygous flies, when stained with the same concentration of RhoVR-Halo (Supplementary Figures 5H–J). However, because the difference in fluorescence intensity in homozygous flies was not significantly larger than heterozygotes, we used heterozygous flies for subsequent experiments.

We established the voltage sensitivity of RhoVR-Halo in fly tissue expressing HaloTag-CD4 using two different approaches. First, we performed electrophysiology using dual two-electrode voltage-clamp combined with fluorescence imaging at the larval Drosophila neuromuscular junction (NMJ). We used the motor neuron driver OK6-GAL4 to drive pre-synaptic expression of HaloTag-CD4 (Figures 7a–f) or the muscle driver G14-GAL4 to express HaloTag-CD4 in the post-synaptic muscle (Figures 7g–l). In live 3rd instar larval NMJ preparations, RhoVR-Halo (2 μM) clearly stains pre-synaptic neuronal compartments when HaloTag-CD4 expression is targeted in motor neurons (red, Figure 7d), co-localizing with the neuronal plasma membrane marker horseradish peroxidase (HRP, gray, Figures 7e,f). In a complementary fashion, when HaloTag-CD4 is expressed in post-synaptic muscle cells, RhoVR-Halo fluorescence (red, Figure 7j) accumulates at NMJs outside of the neuronal membrane outlined by HRP (gray, Figures 7k,l). RhoVR-Halo readily detects excitatory post-synaptic potentials (EPSPs) in muscle cells, confirmed by simultaneous optical imaging and sharp electrode recordings (Figures 7m–o). Importantly, we next used two-electrode voltage-clamp recordings in a semi-dissected larval preparation with muscle HaloTag-CD4 expression (G14-GAL4 > HaloTag-CD4, Figure 7p). This approach demonstrated that depolarizing potentials result in an increase in RhoVR-Halo signal (m6, Figures 7q–s) with an overall voltage sensitivity of approximately 12% ΔF/F per 100 mV (± 0.2%, n = 8), in reasonably close agreement to the value determined in HEK293T cells (14%, Figure 4). Analysis of electrode (Figure 7r) and optical recordings (Figure 7s) show good correspondence. In contrast, no change in fluorescence signals was observed in an adjacent unclamped/unstimulated muscle cell (m7, Figure 7q, gray).

As a second confirmation of voltage sensitivity in fly tissues, we developed a stereotyped stimulation protocol for imaging in fly brain explants. We generated flies that express both HaloTag and the voltage-sensitive fluorescent protein, ArcLight, in PNs (GH146 GAL4, HaloTag/CyO; ArcLight/TM2) for use as an internal positive control. The use of RhoVR-Halo, with excitation and emission profiles in the green/orange region of the visible spectrum, allows for the simultaneous deployment of GFP-based indicators (Deal et al., 2016, 2020), like ArcLight (Jin et al., 2012). Drosophila antennal lobe projection neurons receive input from the olfactory receptor neurons (ORNs) in the antennae (Kazama and Wilson, 2008). As these projection neurons primarily receive cholinergic input from the ORNs (Restifo and White, 1990), we hypothesized that PNs could be readily stimulated with carbachol (CCH), a non-hydrolyzable acetylcholine mimic. We treated ArcLight/HaloTag-CD4 expressing fly brain explants with carbachol (100 μM) and observed robust fluorescence decreases timed to carbachol treatment, indicating a depolarizing membrane potential response to this neurotransmitter analog (Supplementary Figures 6A–D).

Using this robust stimulation protocol in fly brain explants, we next performed two-color voltage imaging with RhoVR-Halo and ArcLight. As before, we loaded RhoVR-Halo (2 μM) in live flies, removed the brains, and imaged the brain explants using epifluorescence microscopy. Excitation provided alternately with blue (475 nm) or green (542 nm) light to excite ArcLight or RhoVR-Halo, respectively, revealed robust fluorescence responses to carbachol (100 μM) treatment (Figure 8). RhoVR-Halo fluorescence increases with carbachol stimulation (Figures 8a–d), corresponding to membrane voltage depolarization and the turn-on response of RhoVR-type indicators (Deal et al., 2016, 2020). In contrast, ArcLight fluorescence decreases with carbachol stimulation (Figure 8e), showing a fluorescence decrease in response to depolarization, consistent with the turn-off response to depolarization for ArcLight indicators (Jin et al., 2012). Importantly, neither RhoVR-Halo nor ArcLight responds to a control experiment that omits carbachol from the perfusion solution (Figures 8c,f). Finally, the chemical-genetic hybrid approach of RhoVR-Halo enables additional controls to be carried out using the same transgenic flies. When HaloTag/ArcLight expressing flies are treated with TMR-Halo and then stimulated with carbachol, there is no response from the voltage-insensitive TMR-Halo (Figures 8c,g), but ArcLight still responds (Figure 8g). Using a “functionally dead” rhodamine dye in this experiment allows for control experiments to be run in the same transgenic animals as the experiments. Similar experiments with inactive mutants of genetically-encoded indicators/actuators (like ArcLight or GCaMP) would require the generation of separate transgenic animals with the inactivating mutation.

To evaluate the ability of RhoVR-Halo to report on physiological stimuli, we probed the response of RhoVR-Halo in ISNs, cells that respond dynamically to changes in osmolarity. Previous studies demonstrated that increases in osmolarity (240–440 mOsm) evoke hyperpolarizing responses in ISNs (Jourjine et al., 2016). Consistent with this, we find that ISNs expressing HaloTag-CD4 (Nanchung-GAL4) and labeled with RhoVR-Halo hyperpolarize upon an increase in osmolarity, as indicated by decreases in RhoVR fluorescence (Figures 9a–c). In fly brains labeled with voltage-insensitive TMR-Halo, we observe no change in fluorescence (Figures 9a–c). In contrast, flies expressing ArcLight in ISNs show fluorescence increases in response to increased osmolarity (Supplementary Figure 7). Two-color voltage imaging alongside ArcLight in flies that express both HaloTag-CD4 and ArcLight in ISNs (Nanchung-GAL4, UAS-HaloTag-CD4/CyO; UAS-ArcLight/TM2) reveals osmolarity-induced decreases in RhoVR fluorescence coupled with increases in ArcLight fluorescence (Figures 9d–f), while control experiments at constant osmolarity show no responses in either ArcLight or RhoVR fluorescence (Figure 9g). Heterozygous flies expressing HaloTag in ISNs and labeled with RhoVR-Halo also respond to changes in osmolarity (Supplementary Figure 8). Taken together, these data establish the utility of RhoVR-Halo for monitoring sensory-induced changes to membrane potential.