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Neural basis for fasting activation of the hypothalamic–pituitary–adrenal axis

Nature volume 620, pages 154–162 (2023)Cite this article

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Abstract

Fasting initiates a multitude of adaptations to allow survival. Activation of the hypothalamic–pituitary–adrenal (HPA) axis and subsequent release of glucocorticoid hormones is a key response that mobilizes fuel stores to meet energy demands1,2,3,4,5. Despite the importance of the HPA axis response, the neural mechanisms that drive its activation during energy deficit are unknown. Here, we show that fasting-activated hypothalamic agouti-related peptide (AgRP)-expressing neurons trigger and are essential for fasting-induced HPA axis activation. AgRP neurons do so through projections to the paraventricular hypothalamus (PVH), where, in a mechanism not previously described for AgRP neurons, they presynaptically inhibit the terminals of tonically active GABAergic afferents from the bed nucleus of the stria terminalis (BNST) that otherwise restrain activity of corticotrophin-releasing hormone (CRH)-expressing neurons. This disinhibition of PVHCrh neurons requires γ-aminobutyric acid (GABA)/GABA-B receptor signalling and potently activates the HPA axis. Notably, stimulation of the HPA axis by AgRP neurons is independent of their induction of hunger, showing that these canonical ‘hunger neurons’ drive many distinctly different adaptations to the fasted state. Together, our findings identify the neural basis for fasting-induced HPA axis activation and uncover a unique means by which AgRP neurons activate downstream neurons: through presynaptic inhibition of GABAergic afferents. Given the potency of this disinhibition of tonically active BNST afferents, other activators of the HPA axis, such as psychological stress, may also work by reducing BNST inhibitory tone onto PVHCrh neurons.

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Fig. 1: AgRP neurons drive the HPA axis during fasting.
Fig. 2: AgRP neurons regulate the HPA axis through projections to the PVH.
Fig. 3: GABA and NPY decrease GABAergic tone onto PVHCrh neurons.
Fig. 4: GABA and NPY are required for AgRP neuron regulation of the HPA axis.
Fig. 5: AgRP neurons presynaptically inhibit tonically active BNSTGABA neuron afferents to PVHCrh neurons.

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Data availability

Fibre photometry data reported in this study are available from: https://research.bidmc.harvard.edu/datashare/DataShareInfo.ASP?Submit=Display&ID=9. Source data are provided with this paper.

Code availability

The custom analysis code used in this study is available on GitHub (https://github.com/AMDouglass/Douglass_Resch_Madara_etal).

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Acknowledgements

We thank M. Andermann, J. Majzoub and the Lowell laboratory for helpful discussions; J. Yu, C. Wu and Y. Li for technical assistance; S. Liberles for providing the NPY-KO mice; and the Brigham Assay Core for running the ACTH radioimmunoassay. The confocal imaging was performed at the BIDMC Confocal Imaging Core. This work was supported by the National Institutes of Health (NIH) (P30DK046200, R01DK122976, R01DK075632, R01DK089044, R01DK111401 and R01DK096010 to B.B.L. and R00HL144923 to J.M.R.). A.M.D. was supported by the Charles A. King Trust Postdoctoral Research Fellowship Program. H.K. was supported by a Brain & Behavior Research Foundation (BBRF) Young Investigator Grant.

Author information

Author notes

  1. Jon M. Resch

    Present address: Department of Neuroscience and Pharmacology, University of Iowa Carver College of Medicine, Iowa City, IA, USA

  2. These authors contributed equally: Amelia M. Douglass, Jon M. Resch, Joseph C. Madara

Authors and Affiliations

  1. Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Amelia M. Douglass, Jon M. Resch, Joseph C. Madara, Hakan Kucukdereli, Zongfang Yang & Bradford B. Lowell

  2. Departments of Brain Sciences and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel

    Ofer Yizhar

  3. Center for Brain Science, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA

    Abhinav Grama & Masahito Yamagata

  4. Program in Neuroscience, Harvard Medical School, Boston, MA, USA

    Bradford B. Lowell

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Contributions

A.M.D., J.M.R. and B.B.L. conceived the study and designed experiments. J.C.M. performed and analysed slice electrophysiology experiments. A.M.D. and H.K. performed and analysed fibre photometry experiments. O.Y. provided the AAV-eOPN3 and guidance on its use. A.G. and M.Y. made the AAV-synaptophysin-GCaMP6f. A.M.D. and J.M.R. performed and analysed all other experiments and prepared the figures. Z.Y. assisted with stereotaxic surgeries. A.M.D., J.M.R. and B.B.L. wrote the manuscript with input from all authors.

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Correspondence to Bradford B. Lowell.

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Extended data figures and tables

Extended Data Fig. 1 Fasting activation of the HPA axis is regulated by leptin but not by the arcuate → PVH melanocortin system.

a, Scheme of the HPA axis. b, Timeline for leptin injection. c, Corticosterone levels were elevated in mice fasted for 24 h compared to fed mice (n = 8 per group. Two-tailed unpaired t-test- ***P = 0.0006). d, Intraperitoneal administration of leptin reduced corticosterone levels in fasted mice (n = 8 vehicle, n = 9 leptin. Two-tailed unpaired t-test- *P = 0.0130). e, Schematic of optogenetic stimulation of AgRP neurons. f, One hour food intake by Agrp-IRES-Cre mice expressing ChR2 or GFP in the arcuate (n = 4 AgRPGFP, n = 6 AgRPChR2. Two-way ANOVA; Sidak’s multiple comparisons test. AgRPGFP ON vs. AgRPChR2 ON- ***P = 0.0008; AgRPChR2 OFF vs. AgRPChR2 ON- ***P = 0.0001). g, Schematic of Cre-dependent AAV-hM4Di-mCherry stereotaxic injection into the PVH of Mc4r-t2A-Cre mice. h, Three hour food intake by Mc4r-t2A-Cre mice expressing hM4Di or mCherry in the PVH (n = 9 MC4RmCherry, n = 6 MC4RhM4Di. Two-way ANOVA; Sidak’s multiple comparisons test. MC4RmCherry CNO vs. MC4RhM4Di CNO- ****P = <0.0001; MC4RhM4Di Vehicle vs. MC4RhM4Di CNO- **P = 0.0015). i, Timeline for chemogenetic inhibition of PVHMc4r neurons. j, Inhibition of PVHMc4r neurons did not affect corticosterone in fed mice (n = 9 MC4RmCherry, n = 6 MC4RhM4Di. Two-tailed unpaired t-test- P = 0.5933). k, Schematic of Cre-dependent AAV-hM3 Dq-mCherry stereotaxic injection into arcuate nucleus of Pomc-IRES-Cre mice. l, Timeline for chemogenetic activation of POMC neurons. m, Activation of POMC neurons did not affect corticosterone in fasted mice (n = 9 POMCmCherry, n = 7 POMChM3Dq. Two-tailed unpaired t-test- P = 0.6736). Scale bar = 200 µm. Data represent mean ± sem.

Source Data

Extended Data Fig. 2 AgRP neuron collaterals and characterization of GABAergic afferents to PVHCrh neurons.

a, Schematic of collateral mapping via injection of retro-AAV-FlpO into Agrp-IRES-Cre::FLTG mice. b, AgRP neuron terminals detected upon injection of retro-AAV-FlpO into individual AgRP neuron efferent sites (denoted by row headings) in Agrp-IRES-Cre::FLTG mice. a.c: anterior commissure, 3V: third ventricle, a.q: aqueduct, s.c.p: superior cerebellar peduncle. c, Schematic of rabies-based collateral mapping. EnvA pseudotyped rabies-∆G-GFP was injected into AgRP neuron terminal areas (PVH example) in Agrp-IRES-Cre mice that were previously injected with AAV-FLEX-TVA-mCherry in the arcuate. d, AgRP neuron terminals detected upon injection of EnvA pseudotyped rabies-∆G-GFP into individual AgRP neuron efferent sites (denoted by row headings) in Agrp-IRES-Cre mice. e, Schematic of monosynaptic rabies mapping from PVHCrh neurons. f-j, Representative images of starter cells and putative afferents containing rabies-GFP labeled neurons in the PVH (f), BNST and mPOA (g), PV (h), LH and anterior arcuate nucleus (i) and DMH and posterior arcuate nucleus (j). PVH: paraventricular hypothalamus, a.c: anterior commissure, BNST: bed nucleus of the stria terminalis, mPOA: medial preoptic area, 3V: Third ventricle, PV: periventricular hypothalamus, LH: lateral hypothalamus, Arc: arcuate nucleus, DMH: dorsomedial hypothalamus. k, Schematic of recordings of BNST→PVH terminals. These mice are those used in Fig. 5j–o. l, BNST→PVH axons reduced activity in response to a looming hand motion stimulus over the animal (n = 3 mice). m, Expression of NPYRs in major PVH neuron cell types derived from single-cell RNA sequencing of PVH neurons43, plotted as average normalized expression level and fraction of cells in each cluster that express the receptor. Scale bar = 200 µm. Data represent mean ± sem.

Extended Data Fig. 3 Schematic of AAV spread in Agrp-IRES-Cre mice, AAV spread and fiber placements in Agrp-IRES-Cre::Crh-IRES-Cre mice and fiber placements in Agrp-IRES-Cre::LSL-ChR2-eYFP mice.

a, Schematics representing AAV spread (shaded regions) for every animal related to experiments in Fig. 1a–c. Each animal is represented by a different colour. b, Representative image of hM3Dq-mCherry expression in AgRP neuron somas in the arcuate nucleus. c, Schematics representing AAV spread (shaded regions) for every animal related to experiments in Fig. 1a,d–e. Each animal is represented by a different colour. d, Representative image of hM4Di-mCherry expression in AgRP neuron somas in the arcuate nucleus. e, Schematics representing AAV spread (shaded regions) and fiber placement (rectangles) for every animal related to experiments in Fig. 1f–i. Each animal is represented by a different colour. See Fig. 1g for a representative histological image. f, Schematics representing AAV spread (shaded regions) and fiber placement (rectangles) for every animal related to experiments in Fig. 1f,l–m. Each animal is represented by a different colour. g, Representative images of GCaMP6s expression in PVHCrh neurons, optic fiber placement in the PVH and hM4Di-mCherry expression in AgRP neuron somas in the arcuate nucleus. h, Schematics representing fiber placements (rectangles) for every animal related to experiments in Fig. 1n–q and Extended Data Fig. 1e,f. Each animal is represented by a different colour. i, Representative image of ChR2-eYFP expression in AgRP neuron somas in the arcuate nucleus and fiber placement. Scale bar = 200 µm. 3V = Third ventricle. The schematics were created using The Mouse Brain in Stereotaxic Coordinates Second Edition (Paxinos and Franklin).

Extended Data Fig. 4 Schematic of AAV spread and fiber placements in Agrp-IRES-Cre mice.

a, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 2a–c. Each animal is represented by a different colour. b, Representative images of ChR2-mCherry expression in AgRP neuron somas in the arcuate nucleus and fiber placement in the BNST. c, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 2a–c. Each animal is represented by a different colour. d, Representative images of ChR2-mCherry expression in AgRP neuron somas in the arcuate nucleus and fiber placement in the PVH. Scale bar = 200 µm. 3V = Third ventricle. a.c. = anterior commisure. The schematics were created using The Mouse Brain in Stereotaxic Coordinates Second Edition (Paxinos and Franklin).

Extended Data Fig. 5 Schematic of AAV spread and fiber placements in Agrp-IRES-Cre mice.

a, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 2a–c. Each animal is represented by a different colour. b, Representative image of ChR2-mCherry expression in AgRP neuron somas in the arcuate nucleus and fiber placement in the LH. Scale bar = 500 µm. c, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 2a–c. Each animal is represented by a different colour. d, Representative images of ChR2-mCherry expression in AgRP neuron somas in the arcuate nucleus and fiber placement in the PBN. Left scale bar = 200 µm, Right scale bar = 500 µm. 3V = Third ventricle. The schematics were created using The Mouse Brain in Stereotaxic Coordinates Second Edition (Paxinos and Franklin).

Extended Data Fig. 6 Schematic of AAV spread and fiber placements in Agrp-IRES-Cre mice.

a, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 2f–h. Each animal is represented by a different colour. b, Representative images of eOpn3-mScarlet expression in AgRP neuron somas in the arcuate nucleus and fiber placement in the BNST. c, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 2f–h. Each animal is represented by a different colour. d, Representative images of eOpn3-mScarlet expression in AgRP neuron somas in the arcuate nucleus and fiber placement in the PVH. e, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 2f–h. Each animal is represented by a different colour. f, Representative images of eOpn3-mScarlet expression in AgRP neuron somas in the arcuate nucleus and fiber placement in the LH. Scale bar = 200 µm. 3V = Third ventricle. a.c. = anterior commisure. The schematics were created using The Mouse Brain in Stereotaxic Coordinates Second Edition (Paxinos and Franklin).

Extended Data Fig. 7 Schematic of AAV spread and fiber placements in Agrp-IRES-Cre::NPY-KO, Agrp-IRES-Cre::vGatlox/lox and Agrp-IRES-Cre::NPY-KO::vGatlox/lox mice.

a, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 4a–c. Each animal is represented by a different colour. b–d, Representative images of ChR2-mCherry expression in AgRP neuron somas and fiber placement in the arcuate nucleus. Scale bar = 200 µm. 3V = Third ventricle. The schematics were created using The Mouse Brain in Stereotaxic Coordinates Second Edition (Paxinos and Franklin).

Extended Data Fig. 8 Schematic of infusion cannula placements in WT mice.

a, Schematics representing infusion cannula placements (rectangles) for every animal related to experiments in Fig. 4d–f. Each animal is represented by a different colour. b–d, Representative images of cannula placement above the PVH. Scale bar = 200 µm. The schematics were created using The Mouse Brain in Stereotaxic Coordinates Second Edition (Paxinos and Franklin).

Extended Data Fig. 9 Schematic of AAV spread and fiber placements in vGAT-IRES-Cre mice.

a, Schematics representing AAV spread (shaded regions) for every animal related to experiments in Fig. 5c–e. Each animal is represented by a different colour. b, Representative image of hM4Di-mCherry expression in BNSTvGAT neuron somas. c, Schematics representing AAV spread (shaded regions) for every animal related to experiments in Fig. 5c–e. Each animal is represented by a different colour. d, Representative image of hM4Di-mCherry expression in LHvGAT neuron somas. e, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 5f–i. Each animal is represented by a different colour. See Fig. 5g for a representative histological image. Scale bar = 200 µm. a.c. = anterior commisure, i.c. = internal capsule. The schematics were created using The Mouse Brain in Stereotaxic Coordinates Second Edition (Paxinos and Franklin).

Extended Data Fig. 10 Schematic of AAV spread and fiber placements in Agrp-IRES-Cre mice, Mc4r-IRES-Cre and Pomc-IRES-Cre mice.

a, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 5j–n and Extended Data Fig. 2k,l. Each animal is represented by a different colour. See Fig. 5k for a representative histological image. b, Schematics representing AAV spread (shaded regions) for every animal related to experiments in Extended Data Fig. 1g-j. Each animal is represented by a different colour. c, Representative image of hM4Di-mCherry expression in PVHMc4r neuron somas. d, Schematics representing AAV spread (shaded regions) for every animal related to experiments in Extended Data Fig. 1k-m. Each animal is represented by a different colour. e, Representative image of hM3Dq-mCherry expression in POMC neuron somas in the arcuate nucleus. Scale bar = 200 µm. 3V = Third ventricle. The schematics were created using The Mouse Brain in Stereotaxic Coordinates Second Edition (Paxinos and Franklin).

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Douglass, A.M., Resch, J.M., Madara, J.C. et al. Neural basis for fasting activation of the hypothalamic–pituitary–adrenal axis. Nature 620, 154–162 (2023). https://doi.org/10.1038/s41586-023-06358-0

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