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A rapidly acting glutamatergic ARC→PVH satiety circuit postsynaptically regulated by α-MSH

Nature Neuroscience volume 20, pages 42–51 (2017)Cite this article

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A Corrigendum to this article was published on 26 July 2017

This article has been updated

Abstract

Arcuate nucleus (ARC) neurons sense the fed or fasted state and regulate hunger. Agouti-related protein (AgRP) neurons in the ARC (ARCAgRP neurons) are stimulated by fasting and, once activated, they rapidly (within minutes) drive hunger. Pro-opiomelanocortin (ARCPOMC) neurons are viewed as the counterpoint to ARCAgRP neurons. They are regulated in an opposite fashion and decrease hunger. However, unlike ARCAgRP neurons, ARCPOMC neurons are extremely slow in affecting hunger (many hours). Thus, a temporally analogous, rapid ARC satiety pathway does not exist or is presently unidentified. Here we show that glutamate-releasing ARC neurons expressing oxytocin receptor, unlike ARCPOMC neurons, rapidly cause satiety when chemo- or optogenetically manipulated. These glutamatergic ARC projections synaptically converge with GABAergic ARCAgRP projections on melanocortin-4 receptor (MC4R)-expressing satiety neurons in the paraventricular hypothalamus (PVHMC4R neurons). Transmission across the ARCGlutamatergic→PVHMC4R synapse is potentiated by the ARCPOMC neuron-derived MC4R agonist, α-melanocyte stimulating hormone (α-MSH). This excitatory ARC→PVH satiety circuit, and its modulation by α-MSH, provides insight into regulation of hunger and satiety.

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Figure 1: Regulation of feeding by ARCPOMC versus ARCVglut2 neurons.
Figure 2: Effects of fasting on ARCVglut2 neurons and their inhibition by ARCAgRP neuron afferents.
Figure 3: ARCVglut2→PVH projections, unlike ARCPOMC→PVH projections, are effective in glutamatergic transmission.
Figure 4: RNA-seq analysis of ARCVglut2 neurons.
Figure 5: Oxtr expression marks ARCGlutamatergic neurons that rapidly promote satiety.
Figure 6: ARCGlutamatergic and ARCOxtr neurons preferentially engage PVHMC4R neurons.
Figure 7: α-MSH postsynaptically increases excitatory input onto PVHMC4R neurons.

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Change history

  • 20 February 2017

    In the version of this article initially published, α-MSH was referred to at the beginning of the fourth paragraph of the introduction as an MC4R antagonist. This should have read agonist. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank members of B.B.L.'s laboratory for discussions. This research was funded by the following NIH grants to B.B.L.: R01 DK075632, R01 DK096010, R01 DK089044, R01 DK111401, P30 DK046200, P30 DK057521; to J.M.R.: F32 DK103387; to J.N.C.: AHA Postdoctoral Fellowship 14POST20100011. Y.M.-C. was supported by a Charles A. King postdoctoral fellowship. Y.L. was supported by an EMBO postdoctoral fellowship.

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Author notes

  1. Henning Fenselau, John N Campbell and Anne M J Verstegen: These authors contributed equally to this work.

Authors and Affiliations

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

    Henning Fenselau, John N Campbell, Anne M J Verstegen, Joseph C Madara, Jie Xu, Bhavik P Shah, Jon M Resch, Zongfang Yang, Yael Mandelblat-Cerf, Yoav Livneh & Bradford B Lowell

  2. Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, USA

    Bradford B Lowell

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Contributions

H.F. and B.B.L. designed the experiments and analyzed data. J.N.C. performed the single-cell RNA-seq experiments with help from Y.M.-C. H.F. and A.M.J.V. conducted the behavioral and histological studies with help from J.M.R. and Z.Y. H.F. performed the electrophysiological experiments with help from J.C.M. and Y.L. J.X. generated Pomc-IRES-cre mice. B.P.S. generated PomcloxP mice. H.F. and B.B.L. wrote the manuscript with comments from all of the authors.

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Correspondence to Bradford B Lowell.

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Integrated supplementary information

Supplementary Figure 1 Validation of hM3Dq expression and function in ARCPOMC neurons; generation and validation of Pomc-IRES-Cre mice.

a, Experimental schematics. b, Expression of AAV-DIO-hM3Dq-mCherry in ARCPOMC neurons in Pomc-Cre mice determined by mCherry immunolabeling (magenta). c, Representative trace of cell-attached recordings from a hM3Dq-expressing ARCPOMC neuron showing an increase in firing rate in response to CNO (10 μM). d, Expression of AAV-DIO-hM3Dq-mCherry (magenta) and Fos (green) in ARCPOMC neurons (right, overlay) in mice sacrificed 90 minutes after CNO administration. e, Effect of CNO/hM3Dq stimulation of ARCPOMC neurons on food intake. Mice from multiple litters, n = 5 animals. Paired two-tailed t-test: 24h: Saline (mean = 4.11, s.e.m. = 0.08843) versus CNO (mean = 3.354, s.e.m. = 0.2454): t(4) = 4.187, *P = 0.0138; 48h: Saline (mean = 7.89, s.e.m. = 0.2434) versus CNO (mean = 6.652, s.e.m. = 0.3109): t(4) = 6.420, **P = 0.0030. Data are presented as mean ± s.e.m. f, Schematic diagram of the Pomc-IRES-Cre allele. The IRES-Cre cassette was targeted just downstream of the stop codon in exon III of the Pomc gene. f, g, tdTomato expression in Pomc-IRES-Cre mice crossed with tdTomato reporter mice. h, tdTomato (magenta) and POMC expression (GFP) in the ARC (right, overlay). Yellow arrows indicate tdTomato-positive/POMC-negative neurons. i, Left, experimental schematic. Right, AAV-DIO-hM3Dq-mCherry expression in ARCPOMC neurons in Pomc-IRES-Cre mice as determined by mCherry immunolabeling. j, Expression of AAV-DIO-hM3Dq-mCherry (left) and Fos (middle) immunolabeling (right, overlay) in ARCPOMC neurons in Pomc-IRES-Cre mice sacrificed 90 minutes after CNO administration.

Supplementary Figure 2 Validation of hM3Dq/hM4Di expression and function in ARCVglut2 neurons.

a, Experimental schematic. b, c, Expression of AAV-DIO-hM3Dq-mCherry (b) or AAV-DIO-hM4Di-mCherry (c) in ARCVglut2 neurons in Vglut2-IRES-Cre mice as determined by mCherry immunolabeling. d, Representative trace of a cell-attached recording from a hM4Di-expressing ARCVglut2 neuron showing a decrease in firing rate in response to CNO (10 μM).

Supplementary Figure 3 Efferent targets of ARCVglut2 neurons.

a, Experimental schematic. AAV-DIO-synaptophysin-mCherry expresses the synaptic vesicle protein, synaptophysin fused with mCherry in a Cre-dependent fashion. b, Expression of AAV-DIO-synaptophysin-mCherry (ARCVglut2 neurons, magenta) and POMC (GFP) immunolabeling in the injection site (ARC). c, d, Expression of AAV-DIO-synaptophysin-mCherry and POMC immunolabeling in the ARC (c) and in the PVH (d). Yellow arrows indicate POMC-positive ARCVglut2 neurons (left) and POMC-positive terminals of ARCVglut2 neurons in the PVH (right). We found that a subset of ARCVglut2 neurons are POMC-positive (c). In contrast, we failed to detect POMC immunolabeling in the vast majority of ARCVglut2 terminals (d) in the PVH. Scale bars represent 20 μm. e, f, POMC (e) or AgRP (f) expression in ARCVglut2 projection sites as determined by expression of AAV-DIO-synaptophysin-mCherry and POMC (e) or AgRP (f) immunolabeling. ARCVglut2 neurons exhibit projections to the bed nucleus of the stria terminalis (BNST), preoptic area (POA), PVH, central nucleus of the amygdala, paraventricular thalamus (PVT), lateral hypothalamic area, dorsomedial hypothalamic nucleus, ventrolateral periaqueductal grey, lateral parabrachial nucleus and nucleus of solitary tractus. Qualitative assessment of fiber density demonstrated that the PVH, PVT, POA and BNST exhibited the most prominently labeled projections from ARCVglut2 neurons.

Supplementary Figure 4 Neuropeptide-defined subtypes of ARCVglut2 neurons and ARCVglut2 single-neuron gene expression of synapse-related genes.

a, Schematic of single-cell RNA-seq clustering analysis based on expression of 90 neuropeptide genes (see Supplemental Table 1). b, Two-dimensional representation of ARCVglut2 neurons (dots) by t-distributed stochastic neighbor embedding (tSNE). Subtypes are annotated according to neuropeptide gene markers. One subtype was not specifically marked by expression of a single neuropeptide (or other) gene and is annotated as “Other.” c, tSNE plot re-colored to show expression of prominent neuropeptide marker genes. Colors of dots indicate the level of expression relative to the maximal level for each gene. c, Expression of synapse-related genes in Pomc-positive (Pomc+) versus Pomc-negative (Pomc-) subsets of ARCVglut2 neurons. Sample size (cells): Pomc-positive/Pomc-negative, n = 10/13. Slc17a6: Pomc-positive (mean = 21.63, s.e.m. = 5.38) versus Pomc-negative (mean = 62.86, s.e.m. = 11.2): Unpaired two-tailed t-test; t(21) = 3.016, **P = 0.0066. Syt1: Pomc-positive (mean = 0.37, s.e.m. = 0.209) versus Pomc-negative (mean = 1.23, s.e.m. = 0.2662): Two-tailed Mann-Whitney test; U = 21, **P = 0.0052. Cplx1: Pomc-positive (mean = 3.412, s.e.m. = 2.054) versus Pomc-negative (mean = 20.26, s.e.m. = 5.574): Two-tailed Mann-Whitney test; U = 20, **P = 0.0041. Data are presented as mean ± s.e.m.

Supplementary Figure 5 ARCKiss1 neurons synaptically engage a small number of PVH neurons and do not influence food intake.

a, Cre-dependent ChR2 fused to a fluorescent protein (AAV-DIO-ChR2-mCherry) was unilaterally targeted to ARCKiss1 neurons. Images show injection site and terminal field in the PVH of ARCKiss1 neurons. Right, schematic shows connection being tested. Representative traces showing assessment of light-evoked EPSCs recorded from an unidentified PVH (uPVH) neuron. b, Expression of AAV-DIO-hM3Dq-mCherry in ARCKiss1 neurons in Kiss1-Cre mice as determined by mCherry immunolabeling. c, Expression of AAV-DIO-hM3Dq-mCherry (left) and Fos (middle) immunolabeling in ARCKiss1 neurons (right, overlay) in mice sacrificed 90 minutes after CNO administration. d, Effect of CNO/hM3Dq stimulation of ARCKiss1 neurons on dark-cycle food intake. Mice from multiple litters, n = 5 animals. Repeated measures two-way ANOVA: Treatment F(1,4) = 0.8651, P = 0.4050; time F(4,16) = 147.8, P < 0.0001; interaction F(4, 12) = 0.4285, P = 0.7860. Data are presented as mean ± s.e.m.

Supplementary Figure 6 CRACM mapping and validation of hM3Dq/ChR2 expression in ARCOxtr neurons.

a, Top, schematic shows connections being tested. Bottom, representative traces showing assessment of light-evoked IPSCs recorded from an uPVH neuron. No PVH-projecting ARCOxtr neurons are GABAergic. b, Expression of AAV-DIO-hM3Dq-mCherry in ARCOxtr neurons as determined by mCherry immunolabeling. c, Expression of AAV-DIO-hM3Dq-mCherry (left) and Fos (middle) immunolabeling in ARCOxtr neurons (right, overlay) in mice sacrificed 90 minutes after CNO administration. d, CNO/hM3Dq stimulation of ARCOxtr reduces refeeding following 24 hours fasting. Mice from multiple litters, n = 5 animals. Repeated measures two-way ANOVA: Treatment F(1,4) = 10.91, P = 0.0298; time F(4,16) = 64.22, P < 0.0001; interaction F(4, 16) = 6.976, P = 0.0019. Sidaks multiple comparisons test: 1h, P = 0.2882; 2h, **P = 0.0018; 3h, ****P = 0.0003; 4h, ****P < 0.0001. Data are presented as mean ± s.e.m. e, Expression of AAV-DIO-ChR2-mCherry (left) in ARCOxtr neurons and placement of optical fiber above the PVH (right).

Supplementary Figure 7 CRACM mapping to evaluate ARC →; PVH connectivity.

a, Left, Cre-independent ChR2 fused to a fluorescent protein (AAV-ChR2-mCherry) was unilaterally targeted to ARC neurons. Images show expression of AAV-ChR2-mCherry in the injection site and terminal field in the PVH. PVHMC4R neurons were transduced with AAV-DIO-GFP. Scale bars represent 100 μm. Right, schematic shows connections being tested (top) and representative traces of light-evoked EPSCs recorded from a PVHMC4R neuron before (black) and after (grey) bath application of the AMPAR antagonist CNQX. ARC, ARCGlutamatergic. b, c, Top, schematics show connections being tested. Bottom, representative traces showing assessment of light-evoked EPSCs recorded from PVHMC4R-negative (MC4R-) neurons.

Supplementary Figure 8 α-MSH effects on electrically evoked EPSCs and schematic diagram of the PomcloxP allele.

a, Left, Representative traces of electrically-evoked AMPAR-mediated EPSCs (black) and NMDAR-mediated EPSCs (grey) recorded under control conditions (top) and with α-MSH treatment (bottom). Right, Bar graph shows the effect of α-MSH on electrically evoked AMPAR/NMDAR ratios. Sample size (cells): Control/α-MSH, n = 5/5. Unpaired two-tailed t-test: Control (mean = 1.077, s.e.m. = 0.2054) versus α-MSH (mean = 2.465, s.e.m. = 0.548): t(8) = 2.373, *P = 0.0451. b, Representative traces of electrically-evoked EPSCs (left) and summary of the effects (right) of α-MSH on paired pulse ratio (PPR). Sample size (cells): Control/α-MSH, n = 9/10. Unpaired two-tailed t-test: Control (mean = 0.9177, s.e.m. = 0.1233) versus α-MSH (mean = 0.9629, s.e.m. = 0.06044): t(17) = 0.3402, P = 0.7379. Recordings were performed from PVHMC4R neurons identified in Mc4r-t2a-Cre::tdTomato mice. 10 slices from 5 mice; mice from multiple litters. Data are presented as mean ± s.e.m. c, Schematic shows the Pomclox allele. Mice with lox sites flanking exon III of the Pomc gene were generated using gene targeting in ES cells.

Supplementary Figure 9 Schematic summarizing the main findings.

Current models for homeostatic control of hunger / satiety lack a rapidly-acting satiety mechanism that is temporally analogous to that provided by rapidly-acting, hunger-promoting ARCAgRP neurons. Our findings report the discovery of this missing component, namely glutamate-releasing oxytocin receptor-expressing neurons in the arcuate nucleus. These neurons converge with ARCAgRP and ARCPOMC neurons on downstream targets and rapidly increase / decrease satiety when experimentally activated / inhibited. Thus, in contrast to the hunger-promoting system where slow- (AgRP) and rapid-acting signals (GABA and NPY) are conveyed by one group of neurons (ARCAgRP neurons), the counteracting satiety-promoting system is split into two parallel-projecting neurons, the previously known ARCPOMC neurons which deliver slow-acting α-MSH, and the previously unknown ARCOxtr neurons which release the rapid-acting transmitter, glutamate. Importantly, these two satiety neurons interact as α-MSH released by ARCPOMC neurons postsynaptically increases glutamatergic transmission across the ARCOxtr→PVHMC4R synapse. This missing rapid-acting component, with ARCPOMC neurons, now provides the full “yang” to the ARCAgRP neuron’s “yin”. Our work also provides a mechanism for α-MSH /MC4R regulation of satiety – plasticity of the novel ARCOxtr→PVHMC4R synapse.

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Fenselau, H., Campbell, J., Verstegen, A. et al. A rapidly acting glutamatergic ARC→PVH satiety circuit postsynaptically regulated by α-MSH. Nat Neurosci 20, 42–51 (2017). https://doi.org/10.1038/nn.4442

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