Deconstruction of a neural circuit for hunger

Abstract

Hunger is a complex behavioural state that elicits intense food seeking and consumption. These behaviours are rapidly recapitulated by activation of starvation-sensitive AGRP neurons, which present an entry point for reverse-engineering neural circuits for hunger. Here we mapped synaptic interactions of AGRP neurons with multiple cell populations in mice and probed the contribution of these distinct circuits to feeding behaviour using optogenetic and pharmacogenetic techniques. An inhibitory circuit with paraventricular hypothalamus (PVH) neurons substantially accounted for acute AGRP neuron-evoked eating, whereas two other prominent circuits were insufficient. Within the PVH, we found that AGRP neurons target and inhibit oxytocin neurons, a small population that is selectively lost in Prader-Willi syndrome, a condition involving insatiable hunger. By developing strategies for evaluating molecularly defined circuits, we show that AGRP neuron suppression of oxytocin neurons is critical for evoked feeding. These experiments reveal a new neural circuit that regulates hunger state and pathways associated with overeating disorders.

Figure 1. ARC^AGRP→ARC^POMC is not required for evoked feeding

a, ARC in Agrp-Cre;Pomc-TopazFP mice expressing ChR2:tdTomato in AGRP neurons. b, Left, scheme for testing ARC^AGRP→ARC^POMC synaptic connections. Red: ChR2:tdTomato. Right, ARC^AGRP→ARC^POMC synaptic currents. Blue: light pulses. c, Synaptic connectivity between AGRP and POMC neurons. Dashed lines: not detected. d, Cell-attached recording (top) and normalised firing rate (bottom, n = 5) from ARC^AGRP→ARC^POMC photostimulation in brain slices (with Npy1r, Npy5r, and GABA_B receptor antagonists). e, POMC neuron expression of hM4D and GFP from Cre-dependent rAAV. f, Top, hM4D agonism with CNO (10 μM). Bottom, firing rate normalised to baseline (paired t-test, n = 4). g, Intraperitoneal CNO (5 mg/kg) did not increase food intake (1 h) in POMC-hM4D mice (paired t-test, P = 0.32, n = 8). h–j, Occlusion of ARC^AGRP→ ARC^POMC inhibition by optical co-stimulation of AGRP and POMC neurons (h) did not impair the feeding response (i,j) (n = 9). Pre, Stim, Post: before, during (blue), after photostimulation (1 h each). Red line: AGRP neuron-evoked food intake from ref. . Values are means ± s.e.m. n.s.: not significant, *P < 0.05.

Figure 2. AGRP axon stimulation evokes feeding in PVH but not PBN

a, Scheme for AGRP axon photostimulation in the PVH and ChR2:tdTomato distribution of bilaterally transduced AGRP axons in the PVH. Dashed line: optical fibre position. b, Food intake before and during photostimulation (1 h each) of AGRP axons in the PVH (n = 16 mice, 8 bilateral and 8 unilateral ChR2 transduction). Red line: somatic AGRP neuron-evoked food intake (ref. 4). c,d, Photostimulation of AGRP axons with a second optical fibre implanted over the PBN (the bilaterally transduced mice used in b). Paired t-test. Values are means ± s.e.m.

Figure 3. Prolonged ARC^AGRP→PVH synaptic inhibition

a, Distribution of ARC^AGRP→PVH postsynaptic neurons. Green: synaptic current detected, red: not detected. Blue tick: light pulse. b, Representative ARC^AGRP→PVH IPSC (above); raster plot of IPSCs (below). c, ARC^AGRP→PVH repetitive photostimulation. Gray: trials, red: average, blue: post-stimulus exponential fit. d, Normalised DC amplitude (see Methods). e,f Post-stimulus delayed release: decay time constant (e) and cumulative charge transfer (f) (sample sizes in parentheses, unpaired t-test). g, Left, PVH neuron silenced by AGRP axon photostimulation (10 Hz, with Npy1r, Npy5r, and GABA_B receptor antagonists). Right, normalised average firing rates (n = 9). Values are means ± s.e.m. *P < 0.05.

Figure 4. PVH neuron inhibition recapitulates feeding from AGRP neuron activation

a, Cre-dependent rAAV expression of hM4D and GFP in PVH SIM1 neurons. b, Food intake before and after intraperitoneal CNO (5 mg/kg) in SIM1-hM4D mouse. c, Food intake before and after CNO (n = 19) or saline (n = 9). Red line: AGRP neuron-evoked feeding (ref. 4). d, Normalised break point for AGRP-ChR2 (n = 6) or SIM1-hM4D (n = 6) mice (2-way ANOVA/1-factor repetition, condition: F_2,20 = 25.5, P <0.001; AGRP/SIM1: F_1,20 = 0.171, P = 0.69; interaction: F_2,20 = 0.45, P = 0.64; Holm-Sidak correction for multiple comparisons). Normalised to food deprivation (dep) break point. e, Scheme for co-photostimulation of AGRP axons and SIM1 neurons over the PVH, and (f) evoked food intake before, during, and after (1 h each) co-stimulation (paired t-test, n = 3). Values are means ± s.e.m. n.s.: not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. ARC^AGRP→PVH^OXT circuit contributes to evoked food intake

a,b, Co-photostimulation scheme for OXT neurons and AGRP axons over the PVH and (b) evoked food intake. Blue shading: photostimulation. c, Disconnection scheme for ARC^AGRP→PVH^OXT. d, Raster plot showing food pellets taken; rows: separate mice. e, Food intake before, during, and after photostimulation (1 h each). Within photostimulation condition: ANOVA, F_3,22 = 18.2, P < 0.001; Holm-Sidak correction for multiple comparisons. Values are means ± s.e.m. n.s.: not significant, *P < 0.05, ***P < 0.001.

Figure 6. Pharmacological dissection of AGRP neuron-evoked feeding

a, Scheme depicting infusion of antagonists (purple) for GABA_A receptors or Npy1r into the PVH followed by ARC^AGRP→PVH axon photostimulation. b, Food intake during photostimulation (1 h) in ChR2-expressing mice (black, n = 5) and ChR2-negative mice (open bars, n = 5). 2-way ANOVA/1-factor repetition; ARC^AGRP→PVH activation: F_1,16 = 24.4, P = 0.001; antagonist: F_2,16 = 13.0, P < 0.001; interaction: F_2,16 = 8.6, P = 0.003. c, As in a except with somatic AGRP neuron activation using hM3D and CNO (0.3 mg/kg). d, Food intake (1 h) after intraperitoneal CNO injection in hM3D-expressing (black, n = 8) and non-transduced control mice (open bars, n = 5). 2-way ANOVA/1-factor repetition; neuron activation: F_1,20 = 22.8, P < 0.001; antagonist: F_2,20 = 14.5, P < 0.001; interaction: F_2,20 = 0.37, P = 0.694. Holm-Sidak correction for multiple comparisons. Values are means ± s.e.m. n.s.: not significant,*P < 0.05, ***P < 0.001