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Hunger enhances food-odour attraction through a neuropeptide Y spotlight

Abstract

Internal state controls olfaction through poorly understood mechanisms. Odours that represent food, mates, competitors and predators activate parallel neural circuits that may be flexibly shaped by physiological need to alter behavioural outcome1. Here we identify a neuronal mechanism by which hunger selectively promotes attraction to food odours over other olfactory cues. Optogenetic activation of hypothalamic agouti-related peptide (AGRP) neurons enhances attraction to food odours but not to pheromones, and branch-specific activation and inhibition reveal a key role for projections to the paraventricular thalamus. Mice that lack neuropeptide Y (NPY) or NPY receptor type 5 (NPY5R) fail to prefer food odours over pheromones after fasting, and hunger-dependent food-odour attraction is restored by cell-specific NPY rescue in AGRP neurons. Furthermore, acute NPY injection immediately rescues food-odour preference without additional training, indicating that NPY is required for reading olfactory circuits during behavioural expression rather than writing olfactory circuits during odour learning. Together, these findings show that food-odour-responsive neurons comprise an olfactory subcircuit that listens to hunger state through thalamic NPY release, and more generally, provide mechanistic insights into how internal state regulates behaviour.

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Fig. 1: Hunger and AGRP neuron activation promote attraction to food odours over pheromones.
Fig. 2: Thalamic AGRP neuron projections promote food-odour attraction.
Fig. 3: NPY and NPY5R are required for hunger-dependent food-odour attraction.

Data availability

All raw data points used for statistical analysis (Prism software) and to generate graphs are reported directly in the figures. Exact P values for pairwise data comparisons in figures, from left to right, using the tests described in the figure legends, are as follows: Fig. 1b: 0.85, 0.002; Fig. 1d: 0.77, 0.002; Fig. 2b (control): 0.82, 0.69, 0.44, 0.56, 0.69, 0.69, 0.69, 0.56, 0.77; Fig. 2b (AGRP-ON): 0.38, 0.69, 0.008, 0.57, >0.99, 0.16, 0.69, 0.31, 0.002; Fig. 2c: <0.0001, 0.0003, 0.0007, 0.0004, 0.29, 0.89, 0.34, 0.59, <0.0001; Fig. 2d: 0.03, 0.84; Fig. 3b (fed): 0.23, 0.38, 0.56; Fig. 3b (fasted): 0.002, 0.002, 0.63; Fig. 3d: 0.30, 0.0005; Fig. 3e: 0.002; 0.85; Fig. 3h: >0.99, 0.03, 0.03; Fig. 3i: 0.03, 0.84. Source data are provided with this paper.

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Acknowledgements

We thank B. Lowell, M. Andermann, S. Datta, M. Albers, J. Flanagan, C. Ran and K. Tao for comments on the manuscript; Q. Li and K. Tao for experimental assistance; and the Nikon Imaging Center at Harvard Medical School for microscopy assistance. The work was supported by a National Institutes of Health grant to S.D.L. (R01 DC013289) and a Uehara Memorial Foundation postdoctoral fellowship and Mishima Kaiun Memorial Foundation funding to N.H. S.D.L. is an investigator of the Howard Hughes Medical Institute.

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N.H. and S.D.L. designed the experiments, N.H. performed the experiments and S.D.L. wrote the manuscript.

Corresponding author

Correspondence to Stephen D. Liberles.

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The authors declare no competing interests.

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Peer review information Nature thanks Michael Krashes and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Controls for two-choice odour-preference assay.

a, Timeline of two-choice behavioural assay. b, c, Odour investigation times of fed (b) and fasted (c) male and female wild-type mice in single odour pairings with water. n (for b) = 10 male mice and 10 female mice; n (for c) = 3 male and 3 female mice. Data are mean ± s.e.m., lines with triangles represent individual mice. *P < 0.05, ***P < 0.001, two-tailed Wilcoxon test (P left to right in b: <0.0001, <0.0001, 0.86; P left to right in c: 0.03, 0.03). A statistical comparison of odour responses in b and c using a two-tailed Mann–Whitney U test revealed significant increases in food-odour investigation in fasted mice compared with fed mice (P = 0.0006) but not in pheromone investigation (P = 0.15). d, Odour investigation times of fed (left) and fasted (right) wild-type female mice. n = 10 mice, data are mean ± s.e.m., lines with triangles represent individual mice. **P < 0.01, two-tailed Wilcoxon test (P fed: 0.77, P fasted: 0.002), e, Odour-investigation times of male (left) and female (right) wild-type mice fed ad libitum. n = 10 mice, data are mean ± s.e.m., lines with triangles represent individual mice, P male, 0.92; P female, 0.19; two-tailed Wilcoxon test. Source data

Extended Data Fig. 2 Mate encounters selectively enhance pheromone attraction in fed mice through an NPY-independent mechanism.

a, b, Timeline of two-choice behavioural assay (a) and odour-investigation times (b) after mate exposure, fed (top) and fasted (bottom), male (left) and female (right), wild-type and Npy-KO mice. n = 6 mice, data are mean ± s.e.m., lines with triangles represent individual mice. *P < 0.05, two-tailed Wilcoxon test. P left to right, fed: 0.03, 0.03, 0.03, 0.03; P left to right, fasted: 0.03, 0.03, 0.03, 0.03. Source data

Extended Data Fig. 3 Controls for experiments involving optogenetic stimulation of AGRP neuron projections to the PVT.

a, Timeline of two-choice assay involving optogenetic stimulation (blue bar) of AGRP neurons. b, Investigation times of fed control (black) and fed AGRP-ON (red) female mice to pheromones and food odours. n = 10 mice, data are mean ± s.e.m., lines with triangles represent individual mice. **P < 0.01, two-tailed Wilcoxon test (P control: 0.32; P AGRP-ON: 0.02). c, Food intake was measured before (Food 1) and after (Food 2) the odour preference assay, as indicated in the timeline, for female mice used in b. n = 10 mice, data are mean ± s.e.m., triangles represent individual mice, ***P < 0.001, Mann–Whitney U-test (P Food 1: 0.49; P Food 2: <0.0001). d, Preference indices were calculated for fed control (unfilled bars, CON) and fed AGRP-ON (filled bars, AGRP) mice after illumination of brain regions indicated (n values are as reported for the same mice in Fig. 2b, c, data are mean ± s.e.m.). e, Food intake before two-choice assay and optogenetic stimulation (Food 1 in timeline) of mice with optic fibres implanted in various brain regions (n values are as reported for the same mice in Fig. 2b, c, data are mean ± s.e.m., P values by Mann–Whitney U test from left to right: 0.47, 0.52, 0.90, 0.94, 0.18, 0.95, 0.81, 0.57, 0.89). f, Timeline of two-choice behavioural assay involving optogenetic stimulation (blue bar) of AGRP neurons in mice fed ad libitum. g, h, AAV-DIO-ChR2 was injected in the arcuate nucleus of wild-type (control) or Agrp-ires-cre (AGRP-ON) mice, and optic fibres were inserted in the PVT. Odour investigation times in the two-choice behavioural assay (g) and post-test food consumption (h, Food 1 from timeline in f) from indicated mice fed ad libitum. n = 6 (control) and 7 (AGRP-ON) mice, data are mean ± s.e.m., lines with triangles represent individual mice. *P < 0.05, **P < 0.01 (P values by two-tailed Wilcoxon test in g: 0.56, 0.02; P value by two-tailed Mann–Whitney U-test in h: 0.0012). Source data

Extended Data Fig. 4 Attraction to a newly learned food odour is enhanced by hunger and optogenetic stimulation.

a, Timeline of two-choice behavioural assay before learning. b, Investigation times of fed (left) and fasted (right) wild-type male mice to pheromones (unfilled bars) and strawberry-gelatin odour (filled bars) before learning. n = 10 mice, data are mean ± s.e.m., lines with triangles represent individual mice. **P < 0.01, two-tailed Wilcoxon test (P fed: 0.002; P fasted: 0.002). c, Timeline of learning paradigm and two-choice behavioural assay after learning. d, Investigation times to pheromones (unfilled bars) and strawberry-gelatin odour (filled bars) after learning in wild-type fed mice, wild-type fasted mice, wild-type fed mice after arcuate nucleus injection of AAV-DIO-ChR2 and PVT illumination (control), and Agrp-ires-cre fed mice after arcuate nucleus injection of AAV-DIO-ChR2 and PVT illumination. n = 6 (wild-type fed, fasted), 4 (control, PVT) and 8 (AGRP-ON, PVT) mice, data are mean ± s.e.m., lines with triangles represent individual mice. *P < 0.05, **P < 0.01, two-tailed Wilcoxon test (P values from left to right: >0.99, 0.03, 0.88, 0.008). Source data

Extended Data Fig. 5 Controls for optogenetic inhibition experiments.

a, AGRP neurons from Agrp-ires-cre;lsl-halorhodopsin (halorhodopsin is expressed as a fusion protein with YFP) or Agrp-ires-cre;lsl-tdTomato mice were dissociated and responses to light (LED-ON) were measured by whole-cell current clamp recordings. Representative examples (left) and the number of spikes measured (right) across cells from different mice before, during and after photostimulation with normalization to the pre-illumination period. n = 6 cells, data are mean ± s.e.m., lines represent individual cells. *P < 0.05 by two-tailed Wilcoxon test (P pink pre versus light: 0.03, P pink light versus post: 0.03, P grey pre versus light: 0.78, P grey light versus post: 0.38). An additional statistical test involving one-way ANOVA with Tukey’s multiple comparisons test revealed similar results (pink one-way ANOVA: P = 0.0003, with Tukey’s multiple comparisons test: pre versus light: P < 0.0001, light versus post: P < 0.001, grey one-way ANOVA: P = 0.53). b, Timeline and schematic based on published brain section images31 of optogenetic inhibition experiments in PVT. c, Investigation times in the two-choice behavioural assay during arcuate nucleus illumination in Agrp-ires-cre (control) or Agrp-ires-cre;lsl-halorhodopsin (ARC-OFF) mice. n = 6 mice, data are mean ± s.e.m., lines with triangles represent individual mice, *P < 0.05 by two-tailed Wilcoxon test (P control: 0.03; P ARC-OFF: 0.44). d, Food consumption after the two-choice odour test (Food 1 in timeline) in Agrp-ires-cre (control) or Agrp-ires-cre;lsl-halorhodopsin (ARC-OFF, PVT-OFF) mice during ARC (left) or PVT (right) illumination. n = 6 mice, mean ± s.e.m., lines with triangles represent individual mice. *P < 0.05, two-tailed Mann–Whitney U-test (P left: 0.015, P right: 0.57). Source data

Extended Data Fig. 6 Food-odour investigation persists beyond transient decreases in AGRP neuron activity, as measured by fibre photometry.

a, Timeline and depiction of fibre photometry experiments during single-odour investigation, with schematic based on published brain section images31. Agrp-ires-cre mice were injected in the arcuate nucleus (ARC) with AAV-DIO-GCaMP6s, and fibre photometry was performed in the ARC, PVT or PVH during water investigation (Test A), pheromone investigation (Test B), food-odour investigation (Test C), and food consumption (Food 1). b, Top row, changes in GCaMP6s fluorescence (ΔF/F) were recorded by fibre photometry in brain regions indicated during single-odour investigation. Responses are depicted as the mean of measurements made in indicated time intervals (0–30 s, 30–60 s or each subsequent minute). n = 6 mice, mean ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001, statistical comparisons between food odour and water responses by two-way ANOVA with Dunnett’s multiple comparison. Second row, food-odour investigation times per 30 s during fibre photometry measurements above at various time intervals (0–30 s, 30–60 s or each subsequent minute). n = 6 mice, data are mean ± s.e.m., lines represent individual mice. Third row, total investigation times for food odour, pheromones and water during 5-min fibre photometry test. n = 6 mice, data are mean ± s.e.m., lines with triangles represent individual mice, *P < 0.05 by two-tailed Wilcoxon test (P water versus pheromone: 0.03; P pheromone versus food odour: 0.03 for ARC, PVT, PVH). Bottom row, changes in GCaMP6s fluorescence (ΔF/F) were recorded by fibre photometry during food consumption (Food 1 in timeline of a) after odour tests in the same mice. Source data

Extended Data Fig. 7 Odour preferences and food consumption patterns in knockout and rescue mice.

a, Timeline of two-choice behavioural assay. b, Investigation times for pheromones and food odours in fed (top) and fasted (bottom) in the indicated female mice. n = 10 mice, data are mean ± s.e.m., lines with triangles represent individual mice. **P < 0.01, two-tailed Wilcoxon test (from left to right, P fed: 0.08, 0.16, 0.24; P fasted: 0.002, 0.002, 0.85). c, Food intake before two-choice assay (Food 1 in timeline) of various knockout male (left) and female (right) mice. n = 10 mice, data are mean ± s.e.m., triangles represent individual mice. d, Cartoon (left) and preference indices (right) of NPY rescue experiments involving Npy-KO (control) and Agrp-ires-cre;Npy-KO (NpyAGRP rescue) mice three weeks after AAV-lsl-Npy injection in the arcuate nucleus. The schematic of the brain is based on published brain section images31. NPY immunohistochemistry is depicted in the figure inset. Preference indices are derived from data in Fig. 3d (n = 12 mice, males and females, mean ± s.e.m. Scale bar, 100 μm). e, Timeline of two-choice behavioural assay for receptor knockouts. f, Investigation times for pheromones and food odours in the fasted female mice indicated. n = 10 mice, data are mean ± s.e.m., lines with triangles represent individual mice. **P < 0.01, two-tailed Wilcoxon test. (P Npy1r-KO: 0.02; P Npy5r-KO: 0.77). g, Food intake after two-choice assay (Food 2 in timeline) of the indicated male and female mice. n = 10 mice, data are mean ± s.e.m., triangles represent individual mice. Source data

Extended Data Fig. 8 Single-odour preference assays in knockout mice.

a, Timeline of behavioural assay. be, Odour investigation times of fed Npy-KO (b), fasted Npy-KO (c), fed Npy5r-KO (d) and fasted Npy5r-KO (e) mice in single-odour pairings with water. n = 3 male mice and 3 female mice, data are mean ± s.e.m., lines with triangles represent individual mice. *P < 0.05, **P < 0.01, two-tailed Wilcoxon test (P values for b: 0.03, 0.001; c: 0.03, 0.03; d: 0.03, 0.03; e: 0.03, 0.03. Source data

Extended Data Fig. 9 Food-search behaviour in knockout mice and analysis of Npy5r expression.

a, Latency to discover a food pellet buried in bedding was timed in the indicated fasted (left) and fed (right) mice. n = 3 male mice and 3 female mice, data are mean ± s.e.m. **P < 0.01, *P < 0.05, Kruskal–Wallis test with Dunn’s multiple comparison. b, RNA in situ hybridization using Npy5r anti-sense and sense probes in coronal brain cryosections with PVT (left, dashed lines) and piriform cortex. Scale bar, 500 μm. c, AGRP neuron projections to the PVT were visualized by immunohistochemistry for tdTomato in Agrp-ires-dre mice injected in the arcuate nucleus with AAV-DIO-tdTomato. Scale bar, 500 μm. Images in b and c are representative of three independent experiments involving different mice. Source data

Extended Data Fig. 10 Controls for NPY injection into PVT.

a, Schematic depicting injection site (top) and preference indices for experiments in Fig. 3h, i. n = 10 male mice and 10 female mice, data are mean + s.e.m. b, Post-test food consumption (Food 1) for mice in Fig. 3h, i. n = 3 male mice and 3 female mice, data are mean ± s.e.m., triangles represent individual mice. *P < 0.05, two-tailed Mann–Whitney U-test (P NPY versus control: 0.015; P NPY5R agonist versus control, 0.04). c, Fasted Npy-KO mice were injected with low NPY levels (0.02 mg kg−1) in the dorsal third ventricle, and after 15 min behaviour was analysed in the two-choice odour preference assay. n = 6 (3 male mice and 3 female mice), data are mean ± s.e.m., lines with triangles represent individual mice. P > 0.99, two-tailed Wilcoxon test. d, Fasted Npy-KO mice were injected with high NPY levels (0.2 mg kg−1) in the ventral third ventricle, and after 60 min behaviour was analysed in the two-choice odour preference assay. n = 20 (10 male mice and 10 female mice), data are mean ± s.e.m., lines with triangles represent individual mice. ***P < 0.001, two-tailed Wilcoxon test. Brain schematics are based on published brain section images31. Source data

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Horio, N., Liberles, S.D. Hunger enhances food-odour attraction through a neuropeptide Y spotlight. Nature 592, 262–266 (2021). https://doi.org/10.1038/s41586-021-03299-4

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