Representations of Taste Modality in the Drosophila Brain

Abstract

Gustatory receptors and peripheral taste cells have been identified in flies and mammals, revealing that sensory cells are tuned to taste modality across species. How taste modalities are processed in higher brain centers to guide feeding decisions is unresolved. Here, we developed a large-scale calcium-imaging approach coupled with cell labeling to examine how different taste modalities are processed in the fly brain. These studies reveal that sweet, bitter, and water sensory cells activate different cell populations throughout the subesophageal zone, with most cells responding to a single taste modality. Pathways for sweet and bitter tastes are segregated from sensory input to motor output, and this segregation is maintained in higher brain areas, including regions implicated in learning and neuromodulation. Our work reveals independent processing of appetitive and aversive tastes, suggesting that flies and mammals use a similar coding strategy to ensure innate responses to salient compounds.

Figure 1. Monitoring activity throughout the entire fly SEZ

A. (left) Schematic of the fly brain showing the SEZ and imaging area (boxed). To monitor activity, 23 1.3µm Z-sections were scanned at 0.5 Hz/Z-stack throughout the entire SEZ of a living fly pre- and post- taste delivery to the proboscis (right). Image of the fly brain was modified from http://www.flybrain.org. B. One Z-section with automated ROIs (outlined in white) marking cell nuclei based on histone-RFP (red) with maximum ΔF response (green) to the 2M sucrose taste stimulus overlaid. Scale is 50 µm. C. Z-section in B, showing cell bodies (green, outlined in white) with maximum ΔF significantly above background (2 st dev) and the nuclear marker, histone-mRFP (red). D. Maximum ΔF of flattened Z-sections representing the entire SEZ, showing activated cell bodies overlaid on average maxF image. E. Schematic representing all responding cells in SEZ (green circles) overlaid on average maxF image. See Movies S1–S4 for raw and processed GCaMP responses and Figure S1 for ΔF responses of single cells and different frame rates.

Figure 2. Sucrose stimulation elicits reproducible and concentration-dependent cell activation in the SEZ

A. Cells responding to 2M sucrose stimulation of the proboscis, with responses to the first (green) and second (red) stimulation overlaid. Images are maximum ΔF of cell bodies in flattened Z-sections (representing anterior 3–12 µm, middle 13–20 µm, and posterior 21–30 µm). Scale is 50 µm. B. Schematic showing all active cells in SEZ, with yellow representing cells activated by two sequential stimulations and red/green by one stimulation for brain in A (brain 1 in C). C. Summary of responding cells for eight brains. Brains 1–5 represent sequential stimulations of 2M sucrose. Brains 6–8 represent one stimulation of 2M sucrose (green) and one stimulation of sweet sensory cells using ATP-induced activation of GR64f cells expressing P2X2 (red) and cells responding to both (yellow). D. Plot of single cell responses (ΔF/F) for two sequential sucrose stimulations for brains 1–5 in C, showing magnitudes of fluorescence changes across trials. E. Stimulation of the fly proboscis with increasing sucrose concentrations (0.01, 0.1, 0.5, 1, 2 M) progressively recruited more taste-responsive cells in the SEZ, shown as schematics of responding cells at the different concentrations. Green denotes cells that responded at the given concentration but not lower concentrations, yellow denotes cells that also responded at a lower concentration. F. The fraction of responding cells by GCaMP calcium imaging and the probability of proboscis extension behavior (PER) showed the same sucrose concentration dependence. Responses are normalized to maximum response. GCaMP (green) is mean ± SEM for 5 brains. Proboscis extension response (red) is 3 trials, n=10 flies/trial, mean ± SEM. Responsiveness in the imaging preparation is reduced as compared to intact flies. See Figure S2 for reproducible activation of central cells to water sensory cell activation.

Figure 3. Reproducibility and dose dependence of bitter responses

A. Cells responding to ATP-induced activation of proboscis GR66a bitter cells expressing the ATP-gated channel P2X2. Responses to the first (green) and second (red) stimulation are overlaid. Images are maximum ΔF of cell bodies (anterior 3–12 µm, middle 13–20 µm, and posterior 21–30 µm). Scale is 50 µm. B. Schematic showing all active cells in SEZ, with yellow representing cells activated by two sequential stimulations and red/green by one stimulation for brain shown in A (brain 1 in C). C. Summary of responding cells for eight brains. Brains 1–5 represent sequential stimulations with a mixture of bitter compounds and ATP. Brains 6–8 represent one stimulation with bitter compounds (green) and one stimulation with ATP for P2X2-mediated activation of GR66a cells (red) and cells responding to both (yellow). D. Plot of single cell responses (ΔF/F) for two sequential bitter stimulations for brains 1–5 in C, showing magnitudes of fluorescence changes across trials. E. Stimulation of the fly proboscis with increasing denatonium concentrations (0.5, 1, 10, 100 mM). Red denotes cells that responded at the given concentration but not lower concentrations, yellow denotes cells that also responded at a lower concentration. Activation of proboscis GR66a bitter cells expressing the ATP-gated channel P2X2 with ATP and a bitter mix activated the most neurons reliably but many of these are also activated by bitter stimuli. F. The number of responding cells by GCaMP calcium imaging increased with concentration. Mean ± SEM for 5 brains.

Figure 4. Taste quality maps in the SEZ

A. Cells responding to ATP-induced activation of proboscis bitter cells expressing P2X2 (red) and cells responding to 2M sucrose (green). Images are maximum ΔF (representing anterior 3–12 µm, middle 13–20 µm, and posterior 21–30 µm). Scale is 50 µm. B. Schematic showing all active cells in SEZ, with red representing cells activated by bitter taste cell activation and green by sucrose stimulation for brain shown in A. Yellow represents cells responding to bitter and sucrose stimulation (brain 1 in C). C. Summary of responding cells for five brains. Cells responding to ATP-induced activation of GR66a bitter cells expressing P2X2 (red), cells responding to 2M sucrose (green) and cells responding to both (yellow). D. Plot of single cell responses (ΔF/F) to bitter stimulations versus sucrose stimulations for brains 1–5 in C, showing modality-selective responses. E. Cells responding to ATP-induced activation of proboscis PPK28 water-sensing cells expressing P2X2 in red and cells responding to 2M sucrose in green. F. Schematic showing all active cells in SEZ, with red representing cells activated by water taste cell activation and green by sucrose stimulation for brain shown in E. Yellow represents cells responding to water and sucrose stimulation (brain 1 in G). G. Summary of responding cells for five brains. Cells responding to ATP-induced activation of PPK28 water-sensing cells expressing P2X2 (red), cells responding to 2M sucrose (green) and cells responding to both (yellow). H. Plot of single cell responses (ΔF/F) to water stimulations versus sucrose stimulations for brains in G. See Figure S3 for a comparison of water versus bitter-responsive cells.

Figure 5. Mixtures do not activate additional cells

A. Schematic showing taste-responsive cells in SEZ, with cells responding to bitter cell activation in red, responding to 2M sucrose stimulation overlaid in green and cells responding to both (yellow) (Separate 1 in C). Scale is 50 µm. B. Schematic showing cells responding to the mixture of bitter cell activation + 2M sucrose, with responding cells color-coded based on the response to single compounds. Cells that responded to the mix only are blue (Mix 1 in C). C. Number of cells responding to individual compounds (Separate) versus the mixture (Mix) for three brains (1,2,3). Color-coding for mixtures is based on the response to single compounds. D. Average number of cells responding to individual compounds (Separate) versus the mixture (Mix), color-coded based on response to single compounds for the 3 brains in C, mean ± SEM, paired t-test, ***p<0.001, **p<0.01. E–H. Comparison of responses to single compounds versus mixtures for 2M sucrose and ATP activation of water sensory cells expressing P2X2, showing a schematic sample brain for single compounds (E), mixture (F), summary graph for 3 brains (G), and average number of cells responding for 3 brains in G, mean ± SEM, paired t-test, *p<0.05 (H). Cells responding to ATP-induced activation of PPK28 water-sensing cells (red), cells responding to 2M sucrose (green), cells responding to both (yellow) and cells responding to mix (blue). I. ΔF/F image of sensory axons responding to 500mM sucrose. J. ΔF/F image of sensory axons responding to 500mM sucrose plus 10mM denatonium. Same brain as I. K. Suppression is concentration-dependent. (left) Fluorescence changes in sensory axons decreased with increasing denatonium concentration. ΔF/F response is normalized to 500mM sucrose response. n=5; mean ± SEM. (middle) Number of sucrose-responsive cells in SEZ decreased as denatonium increased, while number of non-sucrose-responsive cells increased (bitter-responsive cells). n=5; mean ± SEM. (right) Proboscis extension to 500mM sucrose decreased with inclusion of denatonium. 3 trials, n=10 flies/trial, mean ± SEM. See Figure S4 for suppression of sucrose responses in the presence of a GABAB receptor agonist.

Figure 6. Sensory activation elicits motor responses

A. Maximum ΔF of cells responding to sucrose stimulation, with cell bodies overlaid on average maxF image. Scale is 50 µm. (top) Motor neurons filled by photoactivation of C3PA-GFP in proboscis nerves (labelled red). (middle) Overlay showing the sucrose-responsive neurons that are motor neurons (MNs). (bottom). See Figure S5A for a single Z-section. B. Schematic showing all active cells in SEZ, with sucrose-responsive cells not labelled by C3PA-GFP (green), sucrose-responsive motor neurons (yellow), and non-responsive filled motor neurons (red) for brain in A (brain 1 in D). C. Schematic for brain 2 in D. D. Summary of sucrose-responsive cells not labelled by C3PA-GFP (green), sucrose-responsive motor neurons (yellow), and non-responsive filled motor neurons (red) for five brains. E. SEZ taste responses were monitored in DVGLUT-Gal4, UAS-GCaMP6s flies, which express GCaMP6s in glutamatergic motor neurons and interneurons. Brain (brain 1 in G) showing VGLUT cells activated by sucrose stimulation (green) or bitter sensory cell activation (red). F. Second sample brain (brain 2 in G) showing VGLUT cells activated by sucrose stimulation (green) or bitter sensory cell activation (red). The apparent overlap stems from compressing a 3D representation into 2D (the red cells are below the green cells and not-overlapping). G. Summary of VGLUT taste-responsive cells from 5 brains, showing segregation of bitter and sucrose candidate MNs. See Figure S5 for pan-neural calcium imaging with VGLUT cell nuclei labeled, showing activation of VGLUT and non-VGLUT cells.

Figure 7. Sweet and bitter activate different pathways in higher brain regions

A. (left) Schematic of fly brain, showing mushroom bodies (yellow), antennal lobes (blue) and SEZ (green). Image modified from http://www.flybrain.org. (right) Taste-induced activity in the anterior shell of the fly brain, with responses to sucrose sensory stimulation in green and bitter in red. Dotted lines highlight mushroom bodies and antennal lobes (circles). Solid lines outline the central brain, with arrow noting obstruction by the esophagus. Pars intercerebralis is the top group of cells at the midline. Scale is 50 µm. B. Higher resolution view of the upper left quadrant of the fly brain, different brain than A, with sucrose-responsive cell bodies in green and projections in yellow. On right are bitter-responsive cells in red and projections in pink. Scale is 50 µm. C. Comparison of sucrose-responsive (green) and bitter-responsive (red) cell bodies (left) or projections (right). Images are representative of n=5 brains.