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A neural basis for melanocortin-4 receptor–regulated appetite

Abstract

Pro-opiomelanocortin (POMC)- and agouti-related peptide (AgRP)-expressing neurons of the arcuate nucleus of the hypothalamus (ARC) are oppositely regulated by caloric depletion and coordinately stimulate and inhibit homeostatic satiety, respectively. This bimodality is principally underscored by the antagonistic actions of these ligands at downstream melanocortin-4 receptors (MC4R) in the paraventricular nucleus of the hypothalamus (PVH). Although this population is critical to energy balance, the underlying neural circuitry remains unknown. Using mice expressing Cre recombinase in MC4R neurons, we demonstrate bidirectional control of feeding following real-time activation and inhibition of PVHMC4R neurons and further identify these cells as a functional exponent of ARCAgRP neuron–driven hunger. Moreover, we reveal this function to be mediated by a PVHMC4R→lateral parabrachial nucleus (LPBN) pathway. Activation of this circuit encodes positive valence, but only in calorically depleted mice. Thus, the satiating and appetitive nature of PVHMC4R→LPBN neurons supports the principles of drive reduction and highlights this circuit as a promising target for antiobesity drug development.

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Figure 1: PVHMC4R neurons are a downstream target for ARCAgRP-driven hunger.
Figure 2: aBNST and LHMC4R neurons are not a downstream target for ARCAgRP-driven hunger.
Figure 3: PVHMC4R neurons regulate homeostatic satiety in real time.
Figure 4: Glutamatergic PVHMC4R neurons monosynaptically engage the LPBN.
Figure 5: The PVHMC4R→LPBN circuit responds to nutritional state.
Figure 6: The PVHMC4R→LPBN circuit is satiating and appetitive.

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Acknowledgements

The authors gratefully acknowledge the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Mouse Metabolism Core for technical support, V. Petkova and the Beth Israel Deaconess Medical Center Molecular Medicine Core Facility for assistance with quantitative PCR and sample preparation, and D. Morse for the production of the rabies virus. Sequencing and initial data processing were performed at Massachusetts General Hospital's Next-Gen Sequencing Core. Sequencing was supported in part by funding from the Boston Area Diabetes Endocrinology Research Center (BADERC P30 DK057521). This work was supported by the University of Edinburgh Chancellor's Fellowship (A.S.G.); US National Institutes of Health grants to B.B.L. (R01 DK096010, R01 DK089044, R01 DK071051, R01 DK075632, R37 DK053477, BNORC Transgenic Core P30 DK046200, BADERC Transgenic Core P30 DK057521), to M.J.K. (F32 DK089710), to D.P.O. (K08 DK071561) and to J.K.E. (R01 DK088423 and R37 DK0053301); American Heart Association Postdoctoral Fellowship 14POST20100011 to J.N.C.; viral vector production core P30 NS045776 to B.A.T.; and an American Diabetes Association Mentor-Based Fellowship to B.P.S. and B.B.L. This research was supported, in part, by the Intramural Research Program of the NIH, NIDDK (DK075087, DK075088).

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A.S.G., M.J.K., B.P.S. and B.B.L. conceived the studies. A.S.G., M.J.K., C.L. and J.C.M. conducted the studies with assistance from B.P.S., E.W., J.S.S. and D.P.O. Single cell RNA sequencing profiling was conducted by J.N.C. Energy expenditure assays were conducted by O.G. In situ validation of the MC4R-t2a-Cre mouse line was conducted by C.E.L. and J.K.E. Rabies virus was provided by B.A.T. A.S.G., M.J.K. and B.B.L. wrote the manuscript.

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Correspondence to Alastair S Garfield or Michael J Krashes or Bradford B Lowell.

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Integrated supplementary information

Supplementary Figure 1 Central Mc4r-t2a-Cre expression

Mc4r-t2a-Cre expression was demarked by a germline R26-loxSTOPlox-tdTomato reporter allele and assayed across the rostral-caudal extent of the murine neuraxis. The neuroanatomical distribution of MC4R::tdTomato expressing neurons was consistent with the endogenous Mc4r expression profile. Abbreviations: BNST, bed nucleus of the stria terminalis; CeM, central amygdaloid nucleus; DMV, dorsomedial nucleus of the vagus; IML, intermediolateral nucleus; LH, lateral hypothalamus; LPBN, lateral parabrachial nucleus; NTS, nucleus of the solitary tract; PVH, paraventricular nucleus of the hypothalamus; VMH, ventromedial nucleus of the hypothalamus. Scale bar = 100 μm.

Supplementary Figure 2 Validation of PVH Mc4r-t2a-Cre expression

Mc4r-t2a-Cre expression within the paraventricular nucleus of the hypothalamus (PVH) was demarked by injection of a cre-dependent AAV8-hSyn-DIO-GFP viral construct. Colocalization of Mc4r-t2a-Cre::GFP expression with endogenous Mc4r mRNA was determined by way of dual immunohistochemistry (for GFP, brown soma) and radioactive in situ hybridization (for Mc4R mRNA, black puncta). Microscopic analysis revealed extensive colocalisation of the two signals. a, scale bar = 500 μm; b, scale bar = 50 μm; c, scale bar = 10 μm.

Supplementary Figure 3 Neuroanatomical location of optic fibers for in vivo optogenetic occlusion studies

Mice used for optogenetic occlusion studies were validated for fiber placement using histological sections. The approximate positions of the fibers is denoted by an X. a-c, Relates to Figure 1g-h. d, Relates to Figure 2d. e, Relates to Figure 2e. Abbreviations: BNST, bed nucleus of the stria terminalis; LH, lateral hypothalamus; LPBN, lateral parabrachial nucleus; PVH, paraventricular nucleus of the hypothalamus.

Supplementary Figure 4 PVHMC4R neurons do not express oxytocin

Fluorescent immunohistological analysis of MC4R-t2a-Cre::tdTomato (red) and endogenous oxytocin (green) expression in the PVH revealed the complete absence of colocalization at all neuroanatomical levels. Abbreviations: 3v, third ventricle. Scale bar = 100 μm.

Supplementary Figure 5 Validation of in vivo DREADD expression and function

a-d, Validation of excitatory hM3Dq-mCherry expression in Mc4r-t2a-Cre mice. a, Representative image of hM3Dq-mCherry expression within the PVH. b, Membrane potential and firing rate of Mc4r-t2a-Cre::hM3Dq-mCherryPVH neurons increased upon 5 µM CNO application during electrophysiological current clamp recordings. c, Representative image of hM3Dq-mCherry expression within the BNST. d, Representative image of hM3Dq-mCherry expression within the LH. e-h, Validation of inhibitory hM3Dq-mCherry expression in Mc4r-t2a-Cre mice. e, Representative image of hM4Di-mCherry expression within the PVH. f, Membrane potential and firing rate of Mc4r-t2a-Cre::hM4Di-mCherryPVH neurons decreased upon 5 µM CNO application electrophysiological current clamp recordings. g, Representative image of hM4Di-mCherry expression within the BNST. h, Representative image of hM4Di-mCherry expression within the LH. Abbreviations: 3v, third ventricle; aca, anterior commissure anterior part; f, fornix. Scale bar in a, = 100 μm and relates to all images.

Supplementary Figure 6 PVHMC4R neurons do not influence energy expenditure or locomotor activity, while either PVHOXT or PVHCRH neurons do not influence food intake

a-c, Chemogenetic activation of PVHMC4R neurons did not influence a, locomotor activity (LMA; n=11, Paired two-tailed t-test, t(10)=0.43, p=0.67), b, total energy expenditure (TEE; n=11, Paired two-tailed t-test, t(10)=0.20, p=0.85) or c, respiratory exchange ratio (RER; n=11. Paried two-tailed t-test, t(10)=0.35, p=0.79). d-e, PVHOXT and PVHCRH neurons do not influence feeding behavior. d, Chemogenetic inhibition of PVHOXT neurons did not significantly affect light-cycle food intake, compared to the same mice treated with saline (n= n=6, Repeated measures ANOVA, main effect of treatment and interaction not significant, main effect of time (F(4,25)=70.31, p<0.0001). e, Chemogenetic inhibition of PVHCRH neurons did not significantly affect light-cycle food intake, compared to the same mice treated with saline (n=5, Repeated measures ANOVA, main effect of interaction and treatment not significant, main effect of time (F(3,16)=20.61, p<0.0001)..

Supplementary Figure 7 Efferent targets and connectivity of PVHMC4R projections

Neuroanatomical projection mapping from PVHMC4R neurons was achieved via unilateral stereotaxic injection of a synaptically targeted fluorophore (AAV8-hSyn-FLEX-Syn-mCherry). PVHMC4R neurons exhibit exclusively descending and predominantly ipsilateral projections to the median eminence (ME), retrorubral field (RRF), ventrolateral periaqueductal grey (vlPAG), lateral parabrachial nucleus (LPBN), pre-locus coeruleus (pLC), nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus (DMV), rostral ventrolateral medulla (RVLM) and intermediolateral nucleus (IML). Qualitative assessment of fiber density demonstrated that the ME, LPBN and NTS received the densest innervation. CRACM analysis: No light-evoked EPSCs were detected on ARCAgRP neurons, confirming the uni-directionality of the ARCAgRP→PVHMC4R circuit. The vlPAG, pLC and NTS exhibited low to no connectivity, 7%, 0% and 5%, respectively. 56% of LPBN and 55% of DMV of randomly patched post-synaptic neurons exhibited light-evoked EPSCs. Abbreviations: 3v, third ventricle; Aq, aqueduct; cc, central canal; ChAT, choline acetyltransferase; scp, superior cerebellar peduncle; TH, tyrosine hydroxylase.

Supplementary Figure 8 PVHMC4R neurons lie within the ARCPOMC→PVH efferent field

Fluorescent immunohistological analysis of MC4R-t2a-Cre::R26-loxSTOPlox-L10-GFP (green) and endogenous alpha-melanocyte-stimulating hormone (red) expression in the PVH revealed these two fields to be overlapping. Abbreviations: 3v, third ventricle. Scale bar = 100 μm.

Supplementary Figure 9 PVHMC4R→vlPAG or PVHMC4R→NTS/DMV neurons do not influence feeding behavior

a-b, in vivo optogenetic stimulation of PVHMC4R→vlPAG (a; n=4, Repeated measures ANOVA, main effect of interaction and treatment not significant, main effect of time (F(3,12)=76.00, p<0.0001) and PVHMC4R→NTS/DMV (b; n=5, Repeated measures ANOVA, main effect of interaction and treatment not significant, main effect of time (F(3,16)=20.61, p<0.0001) terminals does not promote satiety during dark-cycle feeding. c-e, PVHMC4R→LPBN (c), PVHMC4R→vlPAG (d), PVHMC4R→NTS/DMV (e) mice used for optogenetic feeding and RTPP studies were validated for fiber placement using histological sections. The approximate positions of the fibers is denoted by an X.

Supplementary Figure 10 PVHMC4R→LPBN photostimulation in the absence of ChR2-mCherry does not influence food intake

a, PVHMC4R neurons were transduced with cre-dependent GFP and optic fibers placed bilaterally over the LPBN. b, Photostimulation of PVHMC4R::GFP LPBN terminals had no effect on food intake during the dark-cycle, compared to the same mice without photostimulation (n=10, Repeated measures ANOVA, main effect of treatment and interaction not significant, main effect of time (F(3,36)=95.38, p<0.0001). c, following an overnight fast, as compared to same mice without photostimulation (n=10, Repeated measures ANOVA, main effect of treatment and interaction not significant, main effect of time (n=6, F(3,20)=132.6, p<0.0001). d, PVHMC4R neurons do not make synaptic contact with LPBNMC4R neurons. Channelrhodopsin-assisted circuit mapping between pre-synaptic PVHMC4R neurons (red) and putative post-synaptic LPBNMC4R neurons (green) in an Mc4r-t2a-Cre::R26-loxSTOPlox-L10-GFP mouse line revealed the absence of light-evoked EPSCs in all cells tested.

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Garfield, A., Li, C., Madara, J. et al. A neural basis for melanocortin-4 receptor–regulated appetite. Nat Neurosci 18, 863–871 (2015). https://doi.org/10.1038/nn.4011

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