Graded encoding of food odor value in the Drosophila brain

Abstract

Odors are highly evocative, yet how and where in the brain odors derive meaning remains unknown. Our analysis of the Drosophila brain extends the role of a small number of hunger-sensing neurons to include food-odor value representation. In vivo two-photon calcium imaging shows the amplitude of food odor-evoked activity in neurons expressing Drosophila neuropeptide F (dNPF), the neuropeptide Y homolog, strongly correlates with food-odor attractiveness. Hunger elevates neural and behavioral responses to food odors only, although food odors that elicit attraction in the fed state also evoke heightened dNPF activity in fed flies. Inactivation of a subset of dNPF-expressing neurons or silencing dNPF receptors abolishes food-odor attractiveness, whereas genetically enhanced dNPF activity not only increases food-odor attractiveness but promotes attraction to aversive odors. Varying the amount of presented odor produces matching graded neural and behavioral curves, which can function to predict preference between odors. We thus demonstrate a possible motivationally scaled neural "value signal" accessible from uniquely identifiable cells.

Figure 1.

Odor attraction is graded for differing food odors and increased upon starvation. a, Raw images taken during exposure to yeast odor from fed (top) and starved (bottom) dNPF/OK107;GCaMP3 flies. Red lines are included for quadrant display purposes only and were not present in the behavioral chamber. Odorized quadrant is the right quadrant. b, Example 2D histograms showing cumulative fly counts across the odor chamber for fed (top) and starved (bottom) flies exposed to either no odor (control), a monomolecular odor (acetaldehyde), or food odors. Images reflect the location distributions for the entire 10 min testing period and have been altered to depict the odorized quadrant as the right quadrant. c, From a separate assay, mean percentage of starved flies that ingested each odorized substance when made accessible on dye-suffused filter paper for a 20 min testing period. n = 5 groups of flies per odor. d, Mean percentage of flies in the odorized quadrant for fed (red) and starved (black) flies. ♦p < 0.05, different from attraction to control filtered air. **p < 0.05, fed versus starved. n = 5 groups of flies per odor for each condition. Error bars indicate SEM.

Figure 2.

MB KC odor-associated activity is not related to attraction behavior. a, Schematic of the D. melanogaster olfactory system. MB lobes are omitted for clarity. b, Example mean change in fluorescence (ΔF/F) of the KCs during odor delivery overlaid on the baseline image shows different patterns of activation to two example odors. Inset, Example ΔF/F time course of significantly responding cells to yeast. Blue bar represents the 3 s odor-delivery period. c, Proportion of significantly responding cells is not different for food odors or changed by starvation. d, Mean integrated odor-response (0–5 s after stimulus onset) across significantly responding cells. KC activity does not appear to be related to behavior. n = 4 flies for each condition. Error bars indicate SEM. e, f, Example dendrograms (left) and 3D projections of odor space (right) constructed using multidimensional scaling from a fed (e) and starved (f) fly illustrate that repeated presentations of the same odor cluster together; this is not true of food odors as a category. Sphere size is proportional to the number of significantly responding cells. Sphere colors represent individual odors and relate to colors on dendrogram arms.

Figure 3.

The level of dNPF activity correlates with food-odor attraction. a, Confocal image of dNPF-Gal4/UAS-GCaMP3 (green) with neuropil staining (red) shows the two medial dNPF-positive neurons as clearly visible (downward arrowheads). One of the two lateral dNPF neurons (not imaged in this study) is visible to the left of the image (upward arrow). b, OK107-Gal4;GCaMP3 (green) labels a large number of MB intrinsic neurons, KCs. c, dNPF/mcd8::GFP;MB247DsRED volume reconstruction from a two-photon z-stack in a living fly shows the spatial relationship between the MB (red) and dNPF-Gal4 line (green). Inset, The peduncle of the MB (red) and ipsilateral medial dNPF neuron (green) are visible in the same optical plane in flies expressing dNPF/mcd8::GFP;MB247DsRED transgenes. d, The two structures are easily resolvable for functional imaging using dNPF/OK107;GCaMP3 flies: the dNPF neuron is outlined with a dashed line, and the peduncle is shown with two upward arrowheads. e, Example mean change in fluorescence (ΔF/F) of the dNPF neuron during odor delivery in response to a selection of odors for a fed (top) and starved (bottom) fly. f, Mean ΔF/F time course of the dNPF neuron for fed (red) and starved (black) flies (n = 18 cells from 18 flies for each satiety state) for nine odors. Blue bars represent the 3 s odor-delivery period. g, Mean integrated odor-response (0–5 s after stimulus onset) for all tested odors. The dNPF neuron shows elevated activation to food odors, which are further elevated because of starvation. **p < 0.005, fed versus starved. ♦p < 0.05, different from average response to nonfood odors determined in Figure 1c. Dashed line indicates threshold above which food odors elicit attraction. h, Pearson's r correlation between odor attraction and the mean change in fluorescence (ΔF/F) of the dNPF neuron for all nonfood odors (left, gray) (n = 24; 13 fed, 11 starved) and food odors (right) across satiety states; red represents fed; black represents starved (n = 12; 6 fed, 6 starved). i, Pearson's r correlation between odor attraction and the ΔF/F of the simultaneously imaged peduncle. Error bars indicate SEM.

Figure 4.

The dNPF neuron is necessary to drive food-odor attraction. a, Example 2D histograms showing cumulative fly counts across the odor chamber for starved dNPF/KIR2.1Gal80^ts flies and parental controls tested at 20°C and at 30°C after 3-day 30°C heat shock (gray block) exposed to yeast. Flies show normal attraction to yeast at the permissive temperature and a marked reduction in attraction to yeast at the nonpermissive temperature. Here, and throughout the figure, the left-hand key shows areas of normal activity in blue and disrupted activity in red. Shapes in keys correspond to shapes accompanying 2D histograms. b, Mean yeast attraction for starved dNPF/KIR2.1Gal80^ts flies and parental controls in 20°C and 30°C after heat shock (gray block). After heat shock, dNPF/KIR2.1Gal80^ts attraction to yeast does not differ from attraction to plain air. c, Example 2D histograms for starved dNPF2;hid, dNPF2;hid/cryGal80, and cryGal4/hid flies along with parental controls exposed to yeast. d, Although targeted ablation of the four large dNPF neurons reduces yeast attraction to chance levels, ablation of smaller lateral neurons both dNPF- and cryptochrome-positive does not alter yeast attraction. Yeast remains attractive compared with plain air. e, Example 2D histograms for starved elav/npfr1^dsRNA flies and parental controls exposed to yeast. Pan-neuronal knockdown of dNPF receptors yields a pattern similar to temperature-sensitive selective inactivation and targeted ablation of the four large dNPF cells. f, Mean yeast attraction for starved elav/npfr1^dsRNA flies does not differ from attraction to plain air. ♦p= not significantly different from attraction to control filtered air. n = 5 groups of flies for each condition. Error bars indicate SEM.

Figure 5.

Activating the dNPF neuron is sufficient to produce odor attraction, even to non–food-related odors. a, Example 2D histograms showing cumulative fly counts across the odor chamber for fed UAS-dTRPA1/+;dNPF-Gal4 flies tested in 18°C and 30°C (gray block) exposed to normally attractive (acv), slightly attractive (ea), and aversive (mbut) odors. UAS-dTRPA1/+;dNPF-Gal4 flies show a marked increase in attraction to all odors tested in30°C. b, Mean odor attraction for UAS-dTRPA1/+;dNPF-Gal4 flies in 18°C and 30°C. Activation of the dNPF neuron promotes odor attraction. *p < 0.05, 18°C versus 30°C. Flies carrying multiple copies of the dNPF-Gal4 transgene in combination with UAS-dTRPA1 show elevated levels of odor attraction relative to dNPF-Gal4/UAS-dTRPA1 heterozygous flies. ●p < 0.05, different from dNPF-Gal4/UAS-dTRPA1 heterozygous flies. +, heterozygous for a given transgene; ++, homozygous for a given transgene. n = 10 groups of flies for each condition, fed. Error bars indicate SEM.

Figure 6.

The level of dNPF activity predicts preference when two food odors are in competition. a, Example time courses of the percentage of flies in odorized quadrants for yeast and banana when they are presented simultaneously to separate quadrants measured every 10 s for the 10 min testing period. b, Percentages when yeast and acv, and (c) banana and acv are presented concurrently. d–f, Mean preference for yeast and banana (d), yeast and acv (e), and banana and acv (f) when presented individually versus filtered air or simultaneously to separate quadrants. Data for individual odors versus filtered air were previously shown in Figure 1. ●p < 0.05, different from attraction to odor versus filtered air. n = 5 groups of flies for each condition, starved. g, Mean integrated odor-response of the dNPF neuron (0–5 s after stimulus onset) to a range of concentrations of yeast (green) and a single concentration of acv (orange). n = 10 groups of flies for each condition, starved. h, Corresponding attraction for the same range of concentrations. n = 8 groups of flies for each condition, starved. i, Mean preference for yeast versus acv when varying yeast odor concentration. Yeast concentrations i-iii noted in g. *p < 0.05, different from acv attraction. n = 8 groups of flies for each condition, starved. Error bars indicate SEM. Positions of odorized quadrants in the key are fixed for display purposes only; actual positions were randomized.