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AgRP neurons encode circadian feeding time

Nature Neuroscience volume 27, pages 102–115 (2024)Cite this article

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Abstract

Food intake follows a predictable daily pattern and synchronizes metabolic rhythms. Neurons expressing agouti-related protein (AgRP) read out physiological energetic state and elicit feeding, but the regulation of these neurons across daily timescales is poorly understood. Using a combination of neuron dynamics measurements and timed optogenetic activation in mice, we show that daily AgRP-neuron activity was not fully consistent with existing models of homeostatic regulation. Instead of operating as a ‘deprivation counter’, AgRP-neuron activity primarily followed the circadian rest–activity cycle through a process that required an intact suprachiasmatic nucleus and synchronization by light. Imposing novel feeding patterns through time-restricted food access or periodic AgRP-neuron stimulation was sufficient to resynchronize the daily AgRP-neuron activity rhythm and drive anticipatory-like behavior through a process that required DMHPDYN neurons. These results indicate that AgRP neurons integrate time-of-day information of past feeding experience with current metabolic needs to predict circadian feeding time.

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Fig. 1: ARCAgRP-neuron activity exhibits daily rhythms.
Fig. 2: AgRP-neuron activity rhythm is synchronized to light in free-feeding mice.
Fig. 3: Intact SCN is required for AgRP-neuron activity rhythms in free-feeding mice.
Fig. 4: Impairing input from DMHPDYN neurons attenuates free-feeding AgRP activity rhythms.
Fig. 5: Time-restricted food availability prevails photic cues in determining AgRP-neuron activity rhythms.
Fig. 6: Ablating DMHPDYN neurons impairs AgRP-neuronal and behavioral adaptation to TRF.
Fig. 7: Rhythmic AgRP stimulation partially mimics food entrainment.

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Data availability

Raw data are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

MATLAB and Arduino scripts are available as Supplementary Codes 1–3.

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Acknowledgements

This work is supported by National Institutes of Health grant R01DK126740 to D.A. We thank E. Herzog, S. Sternson and K. Rahmouni for feedback on the paper and J. Resch for providing the 11βHSD2 virus.

Author information

Authors and Affiliations

  1. Department of Neuroscience and Pharmacology, University of Iowa, Iowa City, IA, USA

    Nilufer Sayar-Atasoy, Iltan Aklan, Yavuz Yavuz, Connor Laule, Hyojin Kim, Jacob Rysted, Debbie Davis & Deniz Atasoy

  2. Department of Physiology, School of Medicine, Yeditepe University, Istanbul, Turkey

    Yavuz Yavuz & Bayram Yilmaz

  3. Department of Physiology, School of Medicine, Regenerative and Restorative Medical Research Center (REMER), Istanbul Medipol University, Istanbul, Turkey

    Muhammed Ikbal Alp

  4. Iowa Neuroscience Institute, University of Iowa, Iowa City, IA, USA

    Deniz Atasoy

  5. Fraternal Order of Eagles Diabetes Research Center (FOEDRC), Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA, USA

    Deniz Atasoy

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Contributions

N.S.-A. performed fiber photometry recording experiments and genotyping, prepared MATLAB and Arduino codes for data acquisition, analyzed the data and prepared the figures. I.A. and N.S.-A. performed surgeries. N.S-.A. and I.A. performed optoentrainment experiments. Y.Y., C.L. and I.A. performed electrophysiological recordings. H.K. and J.R. performed imaging. H.K., J.R., I.A. and N.S.-A. contributed to post hoc analysis. D.D. managed mouse breeding. M.I.A. and B.Y. contributed to behavioral setup instrumentation. D.A. and N.S.-A. conceived experiments and prepared the paper.

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Correspondence to Deniz Atasoy.

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Extended data

Extended Data Fig. 1 AgRP-neuron activity shifts to anti-phase in the absence of food.

a. Representative concatenated raw 465 and 405 traces recorded 5 minutes every 30 minutes from AgRP-neurons under AL conditions. Dark shade: dark phase, no shade: light phase. b. Average AgRP activity in dark and light phases (n = 14 mice, two-tailed paired t-test, ***p < 0.0001). c. Left: Averaged daily AgRP GFP activity (n = 5 mice). Right: Rayleigh-plot of AgRP:GFP rhythm in AL fed mice, mean peak: ZT17.7, not significant. d. Daily locomotor activity (normalized to total daily activity of each animal) in LD (left panel) and its heatmap showing each animal’s data (right panel). n = 14 mice. e. Two-tailed Pearson correlation between AgRP-neuron activity and locomotor activity across same circadian time points under AL conditions. f. Representative hourly AgRP GCaMP7s traces (z-score) recorded from mice fasted at ZT11, by acquiring for 10 min at every 30 mins in LD (12 h light:12 h dark). Yellow bar: Light phase, dark gray bar: dark phase. g. Averaged AgRP GCaMP7s rhythms recorded from mice fasted at ZT22 (n = 8 mice). Dashed line is from AL. h. Change in AgRP GCaMP activity in dark and light phases in mice fasted at ZT11 or at ZT22 (two-tailed paired t-test, n = 10 animals, **p = 0.00304, *p = 0.0148) i. Scheme describing the protocol in j. j. Left: AgRP GCaMP activity recorded from 24 h fasted mice that were injected satiety-hormone cocktail during day (ZT3), or night (ZT15). Right: Quantification of activity drop by satiety hormones. k. Two-tailed Pearson correlation between AgRP-neuron activity from mice fasted at ZT22 and previous days’ average food intake across the same circadian timepoints. l. Locomotor activity recorded from mice that were fasted at ZT11 (left panel) and heatmap showing each animal’s data (right panel). n = 10 mice. m. Two-tailed Pearson correlation between AgRP-neuron activity (z-score) and locomotor activity across same circadian time points under fasted conditions (fasting started at ZT11). n,o. Same with l,m, but mice were fasted at ZT22. Data are presented as mean ± SEM. For exact P-values and statistics, see Extended Data Fig. 1 Source Data.

Source data

Extended Data Fig. 2 Effect of light schedule on AgRP-neuron rhythms.

a. Average locomotor activity (left) and food intake (right) rhythms in the first days of constant dark (DD, left, 6 mice). b. Average locomotor activity, food intake, AgRP GCaMP rhythms (5/6 mice were still rhythmic) under AL conditions, Rayleigh-plots (ad lib and fasted phases differ significantly, p = 0.0329, circular one-way ANOVA), and AgRP activity under fasted conditions, respectively, in later days of DD light setting. c. Average locomotor activity (left) and food intake (middle) rhythms in the first days of constant light (LL, 8 mice) and AgRP GCaMP activity on 10th day of LL setting (right). 0/7 animals were rhythmic. d-f. Two-tailed Pearson correlation between average food intake and locomotor activity across same circadian time points under 12 h Light :12 h Dark (d), DD day 9 (e), and LL day 10 (f). g,h. Average AgRP-neuron activity on baseline day (BL, 7 mice) overlaid with the first jetlag day (JD-1, 6 mice) (g) and quantification of post light phase shift mid-dark phase and mid-light phase average AgRP activities (h) in AL fed mice. i,j. Same as (g,h) except no food was provided (8 BL, 6 JD-1 mice). Data are presented as mean ± SEM. For exact P-values and statistics, see Extended Data Fig. 2 Source Data.

Source data

Extended Data Fig. 3 AgRP-neuron specific disruption of cort signaling alters feeding time and meal pattern.

a. Schematic of in vivo fiber photometry recording of AgRP calcium activity in AgRP specific 11βHSD2 expressing (HSD) mice, and representative images showing GCaMP and 11βHSD2 expression in AgRP cells. b,c. Average (b) and individual (heatmap, c) AgRP GCaMP7s activity rhythms recorded from AgRP:HSD mice compared to WT mice in free feeding. d. Rayleigh-plots of AgRP rhythms in HSD animals under AL (mean peak times at ZT4.9, Rayleigh p = 0.0113, Rayleigh test for non-uniformity of circular data) and fasted (mean peak time at ZT23.33, not significant, Rayleigh test for non-uniformity of circular data) conditions. Phases do not differ significantly (one-way circular ANOVA, n = 5 mice each). e. Heatmap showing individual AL locomotor activity in AgRP:HSD mice. f,g. Average (f) and individual (heatmaps, g) daily food intake in AgRP:HSD mice compared to WT mice. Average of 6 days has been shown, n = 5 mice each (two-tailed unpaired t-test, ZT11 *p < 0.05). h,i. Averaged (h) and individual (i) meal size frequencies in wild type and HSD mice (two-tailed unpaired t-test, 6 pellets *p < 0.05). Data are presented as mean ± SEM. For exact P-values, and statistics, see Extended Data Fig. 3 Source Data.

Source data

Extended Data Fig. 4 Circadian variations of synaptic input contribute to AgRP-neuron activity rhythms.

a. Schematic of ex-vivo recording from GFP labeled ARCNPY neurons in Npy-gfp mice and culling times. b,c. Representative loose-seal traces (b, scale: 1 s) and average ARCNPY neuron activity (c) overlaid with average in vivo AgRP:GCaMP7s trace. n = 39-82 neurons for control, and n = 8-19 neurons for data with synaptic blockers. d,e. Representative whole-cell recordings from ARCNPY neurons showing synaptic events (d) and average (e) spontaneous IPSC and EPSC frequency across day overlaid with average in vivo AgRP:GCaMP7s activity. n = 8-17 neurons for sEPSC, and n = 6-22 neurons for sIPSC, two-tailed, unpaired t-test, *p < 0.05, **p < 0.01. Scale: 2 s, 20 pA (sEPSC), 50 pA (sIPSC) f. Schematic of in vivo fiber photometry recording of glutamate dynamics on AgRP-neurons and representative image showing AgRP cells expressing iGluSnFR, and the placement of the fiber in the ARC (Scale 100 μm). g-i. Representative AgRP- iGluSnFR raw trace (g) and averaged signal (h) recorded during access to food in fasted mice and quantification of changes in iGluSnFR signal in the first minutes of food access (two-tailed paired t-test, p = 0.008, n = 3 mice, i). j-l. Summary of changes in glutamate levels on AgRP-neurons over 24 h in AL (average of 3 animals, 3 days, j) and fasted (k) animals, and overlay of two rhythms in averaged in 4 h bins (l, two-tailed paired t-test, *p < 0.05, **p < 0.01). m. Comparison of pre-dark onset (ZT8-ZT12) and dark onset (ZT12-ZT16) levels of glutamate on AgRP-neurons in AL and fasted animals (two-tailed paired t-test, **p = 0.0024, *p = 0.013, n = 3 mice). n-u. Same as f-m, except iGABASnFR was examined (**p = 0.0025, q; **p = 0.0076, *p < 0.05, t; *p = 0.029, u, n = 4 ad lib, n = 5 fasted). Data are presented as mean ± SEM. For exact P-values, number of subjects/neurons, and statistics, see Extended Data Fig. 4 Source Data.

Source data

Extended Data Fig. 5 Impairing input from PVNTRH does not affect rhythms for feeding and AgRP activity.

a. Schematic and representative image of in vivo fiber photometry recording of TRH neuronal Ca2+ activity in PVN. ft: Fiber tract. Scale: 300 μm. b,c. Average PVNTRH Ca2+ activity overlaid with AgRP-neuron activity (from Fig. 1) in AL (b) and fasted (c) states. n = 6 AL, n = 7 fasted animals. d. Rayleigh-plot of PVNTRH activity in AL fed (mean peak time ZT14.7, not significant) and fasted (mean peak time at ZT21, not significant) mice. Phases do not differ significantly. e. Two-tailed Pearson correlation between average fasted PVNTRH and average AgRP activities. f. Schematic of in vivo fiber photometry recording from ARCNPY neurons in mice with PVNTRH ablation. Representative images of ARC:GCaMP7s and PVN showing AAV-tdTomato marking the site of caspase injection (upper scale: 300 μm lower scale: 250 μm). g. Average ARCNPY GCaMP activity in animals with PVNTRH ablation (PVNTRH-X) or with intact PVNTRH neurons (Ctrl). Average of 3 days and n = 4 Ctrl and n = 7 PVNTRH-X mice. h. Rayleigh-plot of ARCNPY GCaMP activity in PVNTRH-X mice (mean peak at ZT7) versus control (mean peak time at ZT7.08). Phases do not differ significantly between two groups. i. Comparison of dark and light phase ARCNPY activity in mice with or without PVNTRH ablation, two-tailed, unpaired t-test, n = 4 ctrl, 7 PVNTRH-X mice. j,k. Feeding pattern of PVNTRH-X mice (j, n = 8 Ctrl and 10 PVNTRH-X mice) and total consumed food during daytime and nighttime (k) compared to control animals, two-tailed, unpaired t-test, n = 8 Ctrl and 10 PVNTRH-X mice. Data are presented as mean ± SEM. For exact P-values and statistics, see Extended Data Fig. 5 Source Data.

Source data

Extended Data Fig. 6 Rhythmic activity of DMHPDYN is altered by food access.

a. Schematic of in vivo fiber photometry recording from ARCNPY neurons in mice whose DMHPDYN neurons have been transduced with AAV-FLEX-hM3D-mCherry. b. Effect of DMHPDYN neuron activation on ARCNPY calcium response (n = 5 mice). c. Average ARCNPY activity in squared period (b) after saline (SAL) or DCZ injection (two-tailed paired t-test, *p = 0.028, n = 5 mice). d. In vivo DMHPDYN neuron activities in AL and fasted mice (6 mice). e. Difference in average DMHPDYN neuron activity in dark (ZT14-20) and light (ZT2-8) phases in AL and fasted mice (n = 6 mice, two-tailed paired t-test, **p = 0.005). f. Schematic and representative image of caspase ablation of DMHPDYN neurons (DMHPDYN-X). g. Circadian amplitude of ARCNPY/AGRP activity in intact (n = 5) and DMHPDYN-X mice (n = 3, two-tailed unpaired t-test, *p = 0.013). h. Schematic and representative image of in vivo fiber photometry recording from ARCNPY neurons in mice with chronic DMHPDYN activation. Scale: 200 μm i. Average ARCNPY activity in mice that received DCZ in their drinking water versus control mice (Ctrl: no DCZ, average of 2 days, n = 5 mice each). j. Rayleigh-plot of ARCNPY activity (DCZ mean peak: ZT5.4, Rayleigh p = 0.0123, Ctrl mean peak: ZT6.4, Rayleigh p = 0.0417, phases do not differ significantly). k. Average ARCNPY activity for dark and light phases, two-tailed, paired t-test, *p = 0.012, n = 5 mice. l. Circadian amplitude of ARCNPY activity in control and DCZ groups (n = 5 mice each). m,n. Daily feeding pattern (m, DCZ vs Ctrl *p < 0.05, **p = 0.0036, 5-days average, n = 6 mice each) and total food intake (n, two-tailed, paired t-test, * p = 0.017, 5-days average, n = 6 mice each). o-r. Same as i,k,m,n, but with mice without viral DREADD injection (n = 4 mice each). s. ARCNPY activity in DCZ group fasted at ZT11 (n = 5 mice). t. Rayleigh-plots showing peak time in fasted control and DCZ mice (Control mean peak: ZT17.81, DCZ: ZT4.04, phases differ significantly, p = 0.0154, circular one-way ANOVA). u. Average dark and light time ARCNPY activity in fasted control and DCZ groups (n = 5 mice, two-tailed, unpaired t-test, *p < 0.03, **p = 0.007. Data are presented as mean ± SEM. For exact P-values, and statistics, see Extended Data Fig. 6 Source Data.

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Extended Data Fig. 7 Time restricted food access synchronizes AgRP-neuron activity rhythms.

a,b. Average daily actogram (a) and average locomotor activity graph on TRF days 23 and no food access day 24 (b) in animals that were given restricted access to food only during daytime (ZT0-12). Orange* denotes fasted days. c. Food intake on day 23 in animals going through daytime TRF. d,e. Nighttime surge in AgRP GCaMP activity dynamics in each mouse shown as heatmap (d) and on average (e) on TRF days 1, 4, 7 and 10 (n = 19, 9, 10, 9 animals, respectively) where animals had access to food between ZT3-7. f. Average locomotor activity in mice under TRF regimen (day 10) compared to AL animals (n = 10 TRF,15 AL mice. Two-tailed unpaired t-test, * p < 0.05, b p < 0.01, a p < 0.001). g. Same as f, but TRF day 11 without food access is compared to fasted animals on otherwise AL schedule (n = 10 mice each). h. Average locomotor activity around food access time at ZT3 in animals on TRF day 10 and day 11 without food access (n = 10 mice, two-tailed paired t-test, ZT3.5 *p = 0.043, ZT4 *p = 0.038). i,j. Two-tailed Pearson correlation of AgRP GCaMP activity (z-score) and locomotor activity in animals on TRF day 10 (i) and day 11 without food access (j). Data are presented as mean ± SEM. for exact P values, see Extended Data 7 Source Data.

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Extended Data Fig. 8 Time restricted food access alters AgRP-neuron activity rhythms.

a. Average locomotor activity in mice under TRF regimen (day-17), where animals had access to food between ZT18-22, compared to their AL days (n = 7 animals, two-tailed paired t-test, *p < 0.05). b. Same as a, but TRF day-18 without food access is compared to fasted days while on AL schedule (*p < 0.05, bp < 0.01, ap < 0.001). c,d. Overlaid average locomotor activities during 24-h (c) and around food access time at ZT18 (d) in animals on TRF with and without food access (n = 7 mice, two-tailed paired t-test, *p < 0.05, bp < 0.01, ap < 0.001). e,f. Two-tailed Pearson correlation of AgRP GCaMP activity and locomotor activity in animals on TRF day-17 (e) and day-18 without food access (f). g-i. AgRP (g,h) and ARCNPY (i) neuron activities recorded from 11βHSD2 expressing (g), SCN-ablated (h) and WT control (i) mice subjected to TRF between ZT3-7 for more than 10 days and was provided no food during the recording period. Upper panels: average of n = 5 (g), n = 8 (h) and n = 4 (i) mice, middle panels: individual data from each animal, lower panels: comparison of AgRP/NPY activity in BL-1 (before light phase, ZT21-0), BL-2 (light onset, before food, ZT0-ZT3) and post-food time (Post or P1, ZT4-7), or P2 (ZT7-10, h) periods. One-way ANOVA followed by Tukey multiple comparison test. *p < 0.05, **p < 0.01 j. AgRP-neuron activity recorded from mice that had been subjected to TRF between ZT18-22 for 17 days. Upper panel: average of n = 6 animals, middle panel: individual data from each animal, lower panel: AgRP activity in BL (ZT15-17.5) and post-food time (ZT19-22) periods ***p < 0.0001. Green shades depict food availability. k-o. AgRP (k-m) and ARCNPY (n,o) neuron activities recorded from 11βHSD2 expressing (k), SCN-ablated (l), SCN-ablated, under DD conditions (m), DMHPDYN-ablated (n) and intact control (o) mice that had been subjected to TRF between ZT3-7 for more than 10 days. Upper panels: average of n = 5 (k,n), n = 8 (l,m) and n = 4 (o) mice; middle and lower panels: same as in g-i, ***p < 0.001). Data are presented as mean ± SEM. For exact P-values, see Extended Data 8 Source Data.

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Extended Data Fig. 9 DMHPDYN neurons provide timed disinhibition to AgRP-neurons in TRF.

a. Left: Schematic of in vivo fiber photometry recording of PDYN neuronal Ca2+ activity in DMH. Right: Diagram describing TRF setting. b,c. Heatmap showing individual (b) and line graph showing averaged (c) DMHPDYN activity in mice under TRF between ZT3-7. n = 6 mice. d. Average DMHPDYN activity in B1 (ZT21-0), B2(ZT0-3) and Post (ZT4-7) periods in TRF and no food access days. Two-way ANOVA with Tukey’s multiple comparison analysis (No food, B1 vs Post, p = 0.012, B2 vs Post p = 0.016, n = 6 mice). e. Change in DMHPDYN activity (Δ = z-score on TRF day – z-score on no food access day from c) overlaid with ARCNPY/AgRP (from Extended Data Figure 8i). Data are presented as mean ± SEM. For exact P values and data, see Extended Data 9 Source Data.

Source data

Extended Data Fig. 10 Allostatic regulation of AgRP-neuron activity by past circadian feeding experience.

a. Proposed model for daily fluctuations in relative AgRP-neuron activity levels. Outside of feeding window, AgRP-neuron activity is relatively unaffected by feeding status (at least on the first day in food deprivation), whereas during feeding time the activity is highly sensitive to food availability. The ‘Adapted feeding window’ is the time of day during which feeding periodically occurred in previous days. In free feeding mice, this window is dark phase and synchronized by light; however, it can be dissociated from circadian phase and reset by restricted food access. b. Schematic depiction of long-term regulation of AgRP-neuron activity by previous day’s circadian feeding experience. Feeding time is encoded into AgRP-neuron activity such that activity rises if anticipated food access does not occur. Top: Under free feeding conditions, AgRP-neuron activity directly or indirectly synchronizes to light through a process that requires intact SCN. Bottom: In time restricted feeding, AgRP activity uncouples from light and synchronizes to feeding time in a process that required intact DMH input.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3 and legends.

Reporting Summary

Supplementary Code 1

MATLAB (2020b) script for analysis of circadian GCaMP data.

Supplementary Code 2

Arduino script for acquiring locomotor activity.

Supplementary Code 3

MATLAB (2020b) script for extracting FED3 data.

Supplementary Data 1

Source data for Supplementary Fig. 3.

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Sayar-Atasoy, N., Aklan, I., Yavuz, Y. et al. AgRP neurons encode circadian feeding time. Nat Neurosci 27, 102–115 (2024). https://doi.org/10.1038/s41593-023-01482-6

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