An excitatory paraventricular nucleus to AgRP neuron circuit that drives hunger

Abstract

Hunger is a hard-wired motivational state essential for survival. Agouti-related peptide (AgRP)-expressing neurons in the arcuate nucleus (ARC) at the base of the hypothalamus are crucial to the control of hunger. They are activated by caloric deficiency and, when naturally or artificially stimulated, they potently induce intense hunger and subsequent food intake. Consistent with their obligatory role in regulating appetite, genetic ablation or chemogenetic inhibition of AgRP neurons decreases feeding. Excitatory input to AgRP neurons is important in caloric-deficiency-induced activation, and is notable for its remarkable degree of caloric-state-dependent synaptic plasticity. Despite the important role of excitatory input, its source(s) has been unknown. Here, through the use of Cre-recombinase-enabled, cell-specific neuron mapping techniques in mice, we have discovered strong excitatory drive that, unexpectedly, emanates from the hypothalamic paraventricular nucleus, specifically from subsets of neurons expressing thyrotropin-releasing hormone (TRH) and pituitary adenylate cyclase-activating polypeptide (PACAP, also known as ADCYAP1). Chemogenetic stimulation of these afferent neurons in sated mice markedly activates AgRP neurons and induces intense feeding. Conversely, acute inhibition in mice with caloric-deficiency-induced hunger decreases feeding. Discovery of these afferent neurons capable of triggering hunger advances understanding of how this intense motivational state is regulated.

Figure 1. Mapping and evaluating connectivity of inputs to AgRP^ARC neurons

a, Rabies schematic. b, EGFP detected in the ARC and DMH (Left) and PVH (Right) in Agrp-ires-Cre mice. c,d, Top, schematic shows connections being tested. Right, representative brains from mice stereotaxically injected with AAV8-DIO-ChR2-mCherry (red = ChR2-mCherry, green = hrGFP from Npy-hrGFP. Bottom, representative traces showing assessment of light-evoked excitatory postsynaptic currents (EPSCs) with blue tic indicating the light pulse (473 nm wavelength, 2 msec). Mice used were Vglut2-ires-Cre; Npy-hrGFP. ChR2 was targeted to (c) DMH and (d) PVH. CNQX = AMPA receptor blocker. e, Average amplitude of light-evoked EPSCs (pA) in AgRP neurons (n = 40 for VGLUT2^DMH→AgRP^ARC group; n = 45 for VGLUT2^PVH→AgRP^ARC group). Results are shown as mean ± SEM, **=p<0.001; see Supplementary Information for statistical analyses. f,g, Representative raster plots of EPSCs for (f) VGLUT2^DMH→AgRP^ARC and (g) VGLUT2^PVH→AgRP^ARC. h,i, Representative traces showing light-evoked changes in membrane potentials in AgRP neurons for (h) VGLUT2^DMH→AgRP^ARC and (i) VGLUT2^PVH→AgRP^ARC. e. n = 45 for VGLUT2^PVH→AgRP^ARC group; n = 40 for VGLUT2^DMH→AgRP^ARC group. Data represent mean ± SEM. P values for unpaired comparisons were calculated by two-tailed Student’s t-test. *p< 0.05, **p <0.001.

Figure 2. TRH^PVH and PACAP^PVH neurons provide excitatory input to AgRP neurons

a-d, Top, schematic shows connections being tested. Right, representative brain sections of PVH injected with AAV8-DIO-ChR2-mCherry (white = ChR2-mCherry). Bottom, representative traces showing assessment of light-evoked EPSCs with blue tic indicating the light pulse (473 nm wavelength, 2 msec). Mice used were (a) Sim1-Cre; Npy-hrGFP, (b) Pdyn-ires-Cre; Npy-hrGFP, (c) Trh-ires-Cre; Npy-hrGFP, and (d) Pacap-ires-Cre; Npy-hrGFP. CNQX = AMPA receptor blocker. e-h, Baseline and effects of PACAP (100 nM) with or without PAC1R blocker (PACAP_6-38; 200 nM) on membrane potential and firing rate of AgRP neurons (n = 18, n = 10, n = 8, respectively). Results are shown as mean ± SEM, **=p<0.001; see Supplementary Information for statistical analyses. g, h. n = 18 for baseline group; n = 10 for PACAP_1-38 group; n = 8 for PACAP_1-38 + PACAP_6-38 group. P values for pair-wise comparisons (baseline versus PACAP_1-38 group) and unpaired comparisons (baseline versus PACAP_1-38 + PACAP_6-38 group) were calculated by two-tailed Student’s t-test. *p< 0.05, **p <0.001.

Figure 3. Fidelity of TRH^PVH/PACAP^PVH→ARC and AgRP^ARC→PVH circuitry

a-f, Left, schematic shows connections being tested. Right, representative traces showing assessment of light-evoked EPSCs (a-c) or IPSCs (d-f) with blue tic indicating the light pulse (473 nm wavelength, 2 msec). Mice used were (a) Trh-ires-Cre; Pomc-hrGFP, (b-c) Pacap-ires-Cre; Pomc-hrGFP, (d) Agrp-ires-Cre; Sim1-Cre; R26-loxSTOPlox-L10-GFP mice for visualization of SIM1 neurons, (e) Agrp-ires-Cre; Trh-ires-Cre injected with AAV8-DIO-mCherry into the PVH for visualization of TRH neurons and (f) Agrp-ires-Cre; Pacap-ires-Cre; R26-loxSTOPlox-L10-GFP mice for visualization of PACAP neurons. CNQX = AMPA receptor blocker. PTX = GABA_A receptor blocker. (g) Model summarizing reciprocal circuitry and its fidelity.

Figure 4. DREADD-mediated manipulation of TRH^PVH or PACAP^PVH neurons mediates feeding through AgRP neurons

a,d, Membrane potential and firing rate of (a) TRH^PVH or (d) PACAP^PVH neurons transduced with AAV8-DIO-hM3Dq-mCherry upon CNO application. b,e, AAV8-DIO-hM3Dq-mCherry was transduced unilaterally into the PVH of (b) Trh-ires-Cre or (e) Pacap-ires-Cre mice and ipsilateral induction of Fos (red) was assessed in AgRP neurons (marked by hrGFP, green) 3 hrs following CNO (0.3 mg kg^-1) injection (n=3, n=3; respectively). c,f, Left, AAV8-DIO-hM3Dq-mCherry (white) was transduced bilaterally into the PVH of (c) Trh-ires-Cre or (f) Pacap-ires-Cre mice. Right, light-cycle food intake after injection of saline (black) or CNO (red; 0.3 mg kg^-1) (n=7-8 animals per condition; experiment replicated three times per animal). g,h, Simultaneous inhibition of AgRP^ARC neurons with activation of TRH^PVH neurons attenuates food intake. (g) Sagittal schematic of occlusion study, whereby AAV8-DIO-hM3Dq-mCherry (white) was transduced bilaterally into the PVH (Top) and AAV8-DIO-hM4Di-mCherry (white) was transduced bilaterally into the ARC (Bottom) of Trh-ires-Cre; Agrp-ires-Cre mice. (h) light-cycle food intake after injection of saline (black) or CNO (red; 0.3 mg kg^-1) in Trh-ires-Cre; Agrp-ires-Cre (dotted; hM3Dq in PVH and hM4Di in ARC) and Trh-ires-Cre (solid; hM3Dq in PVH) mice (n=4 animals per condition; experiment replicated three times per animal). i, (Top) Membrane potential and firing rate of TRH^PVH neurons transduced with AAV8-DIO-hM4Di-mCherry upon CNO application. (Bottom) AAV8-DIO-hM4Di-mCherry (white) was transduced bilaterally into the PVH of Trh-ires-Cre mice. j, Dark-cycle food intake after injection of saline (black) or CNO (red; 0.3 mg kg^-1) (n=4 animals per condition; experiment replicated three times per animal). Results are shown as mean ± SEM, *=p<0.05, **=p<0.001; see Supplementary Information for statistical analyses. b,e. n = 3 for each group. Data represent mean ± SEM. P values for unpaired comparisons were calculated by two-tailed Student’s t-test. *p< 0.05. c, f. n = 8 for Trh-ires-Cre group; n = 7 for Pacap-ires-Cre group. Data represent mean ± SEM. Two-way repeated measures ANOVA detected significant interaction of ‘time’ and ‘treatment’. F_3,20 = 56.13, **p <0.001; F_3,18 = 0.056, **p <0.001; respectively. Sidak’s post-hoc test shows significant difference between ‘time’ and ‘treatment’ at 1 and 2 hrs as indicated; respectively. h. n = 4 for each group. Data represent mean ± SEM. Two-way repeated measures ANOVA detected significant interaction of ‘genotype’ and ‘treatment’. F_3,18 = 6.083, *p < 0.05. Sidak’s post-hoc test shows significant difference between ‘genotype’ and ‘treatment’ as indicated. j. n = 4. Data represent mean ± SEM. Two-way repeated measures ANOVA detected significant interaction of ‘time’ and ‘treatment’. F_3,18 = 3.962, *p < 0.05. Sidak’s post-hoc test shows significant difference between ‘time’ and ‘treatment’ at 3 hrs as indicated.