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

Abstract

Arcuate nucleus (ARC) neurons sense the fed or fasted state and regulate hunger. Agouti-related protein (AgRP) neurons in the ARC (ARC^AgRP neurons) are stimulated by fasting and, once activated, they rapidly (within minutes) drive hunger. Pro-opiomelanocortin (ARC^POMC) neurons are viewed as the counterpoint to ARC^AgRP neurons. They are regulated in an opposite fashion and decrease hunger. However, unlike ARC^AgRP neurons, ARC^POMC 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 ARC^POMC neurons, rapidly cause satiety when chemo- or optogenetically manipulated. These glutamatergic ARC projections synaptically converge with GABAergic ARC^AgRP projections on melanocortin-4 receptor (MC4R)-expressing satiety neurons in the paraventricular hypothalamus (PVH^MC4R neurons). Transmission across the ARC^{Glutamatergic}→PVH^MC4R synapse is potentiated by the ARC^POMC 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.

Figure 1. Regulation of feeding by ARC^POMC versus ARC^Vglut2 neurons

a, Experimental schematic. b, d, e, Effect of CNO/hM3Dq stimulation of ARC^POMC neurons (b, d) or ARC^Vglut2 neurons (e) on dark-cycle feeding. c, Effect of CNO/hM3Dq stimulation of ARC^POMC neurons on iBAT temperature. f, Effect of CNO/hM4Di inhibition of ARC^Vglut2 neurons on light-cycle feeding. b–f, Data are presented as mean ± SEM. Mice from multiple litters. Repeated measures two-way ANOVA followed by Sidak’s multiple comparisons test (b, n = 5 animals; Treatment F(1,4) = 0.3265, P = 0.5983; time F(4,16) = 89.82, P < 0.0001; interaction F(4,16) = 3.037, P = 0.0485. 1h, P = 0.4831; 2h, P = 0.9980; 3h, P = 0.4831; 4h, P = 0.0610; c, n = 4 animals. Treatment F(1,3) = 8.931, P = 0.0582; time F(5,15) = 8.008, P = 0.0008; interaction F(5, 15) = 6.494, P = 0.0021. 30 min, ****P < 0.0001; 1h, ****P < 0.0001; 2h, **P = 0.0011; 3h, *P = 0.0102; 4h, ***P = 0.0008; d, n = 4 animals; Treatment F(1,3) = 0.7301, P = 0.4557; time F(4,12) = 75.69, P < 0.0001; interaction F(4, 12) = 1.475, P = 0.2703; e, n = 4 animals. Treatment F(1,3) = 63.28, P = 0.0041; time F(4,12) = 40.61, P < 0.0001; interaction F(4, 12) = 10.40, P = 0.0007. 1h, **P = 0.0040; 2h, ****P < 0.0001; 3h, ****P < 0.0001; 4h, ****P < 0.0001; f, n = 6 animals. Treatment F(1,5) = 16.55, P = 0.0096; time F(4,20) = 24.66, P < 0.0001; interaction F(4, 20) = 7.808, P = 0.0006. 1h, ***P = 0.0002; 2h, **P = 0.0016; 3h, ****P < 0.0001; 4h, ****P < 0.0001).

Figure 2. Effects of fasting on ARC^Vglut2 neurons and their inhibition by ARC^AgRP neuron afferents

a, Top, schematic shows experimental approach used in (a) and (b). a, Representative traces (middle) and summary (bottom) of fasting effects on the firing rate of ARC^Vglut2 neurons as assessed using cell-attached recordings. Number of animals: Fed/fasted, N = 3/3. Sample size (cells): Fed/fasted, n = 19/17; Fed (M = 0.7437, s.d. = 0.6771) versus fasted (M = 0.2208, s.d. = 0.4725): Two-tailed Mann-Whitney test: U = 61, ***P = 0.0007. b, Representative traces (top) and summary (bottom) of fasting effects on sIPSCs in ARC^Vglut2 neurons. Number of animals, fed/fasted, N = 3/3; sample size (cells), fed/fasted, n = 12/13. Unpaired two-tailed t-test: sIPSC amplitude: Fed (M = 37.33, s.d. = 12.95) versus fasted (M = 51.34, s.d. = 15.5): t(23) = 2.442, *P = 0.0227; sIPSC frequency: Fed (M = 0.9563, s.d. = 0.6858) versus fasted (M = 2.423, s.d. = 1.667): t(23) = 2.830, **P = 0.0095. Data are presented as mean ± SEM. c, ARC^AgRP→ARC^Vglut2 CRACM. Top, schematic of the connection tested. ARC^AgRP and ARC^Vglut2 neurons were transduced with a Cre-dependent ChR2 tagged with mCherry (AAV-DIO-ChR2-mCherry). ChR2-expressing ARC^AgRP neurons were identified by co-expression of mCherry and hrGFP (note that NPY expression in the ARC marks all AgRP neurons). ARC^Vglut2 neurons were identified by expression of mCherry and absence of hrGFP. Bottom, representative traces of light-evoked IPSCs before and after bath application of bicuculline (GABA_A receptor antagonist). V_h = 0 mV was used to prevent movement of Na⁺ and Ca²⁺ through ChR2 which was expressed in the patched ARC^Vglut2 neurons, in addition to the afferent ARC^AgRP neurons.

Figure 3. ARC^Vglut2→PVH projections, unlike ARC^POMC→PVH projections, are effective in glutamatergic transmission

a–c, ARC→PVH CRACM. Top, schematics showing connection being tested. ARC^POMC or ARC^Vglut2 neurons were transduced with Cre-dependent ChR2. Below, representative traces showing assessment of light-evoked EPSCs. c, Bottom, representative traces of light-evoked EPSCs before and after bath application of CNQX (AMPAR antagonist)

Figure 4. RNA-seq analysis of ARC^Vglut2 neurons

a, Schematic and representative ARC dissection images for single-cell RNA-seq of GFP-positive ARC^Vglut2 neurons. Scale bar represents 200 μm and applies to all images. b, Detection of housekeeping (Gapdh), glutamatergic (Slc17a6; Vglut2), GABAergic (Slc32a1; Vgat), and selected neuropeptide (Pomc, Cartpt, Kiss1, and Tac2) transcripts in ARC^Vglut2 neurons. Expression values are in units of RPKM (reads per kilobase gene model per million mapped reads). c, Percentages of ARC^Vglut2 neurons that are Pomc/Cartpt, Pomc only, Kiss1/Tac2 or “Other” neurons. d, Co-localization of POMC and GFP immunoreactivity in the ARC from a Vglut2-IRES-Cre::L10-GFP mouse. Scale bar represents 100 μm and applies to all images.

Figure 5. Oxtr expression marks ARC^{Glutamatergic} neurons that rapidly promote satiety

a, Oxtr expression in ARC^Vglut2 neurons as determined by RNA-seq. b, Co-localization of POMC protein in tdTomato-positive ARC^Oxtr neurons. Approximately 50% of ARC^Oxtr neurons expressed POMC. c, Representative trace (top) and summary (bottom) of oxytocin’s effect on ARC^Oxtr neuron firing rate. Scale bars, 50 pA, 30 sec. 6 slices from 3 mice; mice from multiple litters; cells, n = 6. Baseline (M = 1.261, s.d. = 0.8192) versus Oxytocin (M = 2.991, s.d. = 1.495): Paired two-tailed t-test, t(5) = 4.253, **P = 0.0081. d, CRACM of ARC^Oxtr→PVH connectivity. Below, representative traces showing light-evoked EPSCs. e, Effect of CNO/hM3Dq stimulation of ARC^Oxtr neurons on dark-cycle feeding (n = 6 animals). f, Left, experimental schematic. Right, effect of ARC^Oxtr terminal photostimulation in the PVH on dark-cycle feeding (n = 5 animals). e, f, Data are presented as mean ± SEM. Repeated measures two-way ANOVA followed by Sidak’s multiple comparisons test. e, Mice from multiple litters, n = 6 animals. Treatment F(1,5) = 26.25, P = 0.0037; time F(4,20) = 63.88, P < 0.0001; interaction F(4, 20) = 15.31, P < 0.0001.1h, P = 0.1760; 2h, **P = 0.0025; 3h, ****P < 0.0001; 4h, ****P < 0.0001. f, Mice from multiple litters, n = 4 animals. Repeated measures two-way ANOVA: Treatment F(1,3) = 36.87, P = 0.0090; time F(4,12) = 24.90, P < 0.0001; interaction F(4, 12) = 14.04, P = 0.0002. Sidaks multiple comparisons test: 1h, P = 0.1276; 2h, *P = 0.0195; 3h, ***P = 0.0001; 4h, ****P < 0.0001.

Figure 6. ARC^{Glutamatergic} and ARC^Oxtr neurons preferentially engage PVH^MC4R neurons

a–c, CRACM of ARC→PVH connectivity. Top, schematics show connections being tested. Below, representative traces showing assessment of light-evoked EPSCs. d, Percentage of PVH^MC4R+ (MC4R-positive) or PVH^MC4R− (MC4R-negative) neurons with light-evoked EPSCs. e, Convergent CRACM of ARC^AgRP and ARC^Oxtr afferents onto PVH neurons. Schematic shown on the top right. Left, representative traces of light-evoked ARC^AgRP-mediated IPSCs (top)/ARC^Oxtr-mediated EPSCs (bottom) recorded from the same uPVH neuron. Right, Venn diagram summary for all 10 neurons receiving input.

Figure 7. α-MSH post-synaptically increases excitatory input onto PVH^MC4R neurons

a, Top, experimental schematic. a, b, Representative traces (a, bottom) and summary (b) of α-MSH effects on sEPSCs. 8 slices from 4 mice; mice from multiple litters; sample size (cells): control/α-MSH, n = 12/13. sEPSC amplitude: Control (M = 17.6, s.d. = 3.923) versus α-MSH (M = 25.88, s.d. = 7.668): Unpaired two-tailed t-test, t(23) = 3.353, **P = 0.0028. sEPSC frequency: Control (M = 1.29, s.d. = 1.011) versus α-MSH (M = 1.379, s.d. = 0.8736): Two-tailed Mann-Whitney test: U = 65, P = 0.4951 b, Bottom, sEPSC amplitude distribution showing that α-MSH significantly increased the frequency of large-amplitude sEPSCs (Kolmogorov-Smirnov test, P < 0.0001). c, Left, experimental schematic. Right, POMC and mCherry immunolabeling in control and Pomc knockout (Pomc KO) mice. c, d, Representative traces (c, Bottom) and summary (d) of Pomc KO effects on sEPSCs. Number of slices: Control/Pomc KO, N = 4/4. Number of animals: Control/Pomc KO, N = 2/2. Sample size (cells): Control/Pomc KO, n = 8/5; sEPSC amplitude: Control (M = 18.96, s.d. = 5.721) versus Pomc KO (M = 12.88, s.d. = 1.293): Unpaired two-tailed t-test, t(11)= 2.304, *P = 0.0417. sEPSC frequency: Control (M = 1.703, s.d. = 0.853) versus Pomc KO (M = 1.553, s.d. 0.3897): Unpaired two-tailed t-test: t(11) = 0.3653, P = 0.7218. Bottom, sEPSC distribution showing decreased frequency of large-amplitude sEPSCs in Pomc KO mice (Kolmogorov-Smirnov test, P < 0.0001). e, Experimental schematic. f, g, Representative traces (left) and summary (right) of α-MSH effects on light-evoked AMPAR/NMDAR ratios (f, slices from 3 mice; mice from multiple litters. Sample size (cells): Control/α-MSH, n = 8/11; Control (M = 3.365, s.d. = 1.622) versus α-MSH (M = 5.642, s.d. = 1.980): Unpaired two-tailed t-test: t(17) = 2.661, *P = 0.0165) and on light-evoked Paired-Pulse ratios (PPRs) (g, Slices from 3 mice; mice from multiple litters. Sample size (cells): control/α-MSH, n = 4/6. Control (M = 0.72, s.d. = 0.1186) versus α-MSH (M = 0.6533, s.d. = 0.1093): Unpaired two-tailed t-test: t(8) = 0.9150, P = 0.387).