Published: 02 February 2014

An excitatory paraventricular nucleus to AgRP neuron circuit that drives hunger

Michael J. Krashes¹^na1^nAff7,
Bhavik P. Shah¹^na1^nAff7,
Joseph C. Madara¹,
David P. Olson¹^nAff7,
David E. Strochlic^2,3,
Alastair S. Garfield^1,4,
Linh Vong¹^nAff7,
Hongjuan Pei⁵,
Mitsuko Watabe-Uchida⁶,
Naoshige Uchida^3,6,
Stephen D. Liberles^2,3 &
…
Bradford B. Lowell^1,3

Nature volume 507, pages 238–242 (2014)Cite this article

34k Accesses
443 Citations
126 Altmetric
Metrics details

Subjects

Hypothalamus

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^1,2,3,4,5. Consistent with their obligatory role in regulating appetite, genetic ablation or chemogenetic inhibition of AgRP neurons decreases feeding^3,6,7. 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^8,9,10. 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.

You have full access to this article via your institution.

Reciprocal activity of AgRP and POMC neurons governs coordinated control of feeding and metabolism

Article Open access 20 February 2024

Deciphering an AgRP-serotoninergic neural circuit in distinct control of energy metabolism from feeding

Article Open access 10 June 2021

TrkB-expressing paraventricular hypothalamic neurons suppress appetite through multiple neurocircuits

Article Open access 07 April 2020

Main

To identify monosynaptic inputs to AgRP neurons, we used a modified rabies virus SADΔG–EGFP (EnvA)¹¹ in combination with Cre-dependent helper adeno-associated viruses (AAVs) expressing TVA (receptor for the avian sarcoma leucosis virus glycoprotein EnvA; AAV8-FLEX-TVA–mCherry) and RG (rabies envelope glycoprotein; AAV8-FLEX–RG). When used with Agrp-IRES-Cre mice, TVA and RG, respectively, allow for rabies infection of AgRP neurons and subsequent retrograde transynaptic spread^11,12 (Fig. 1a). AAV targeting of the helper viruses was specific to AgRP neurons (Supplementary Fig. 1). Three weeks post-AAV transduction, we injected SADΔG–EGFP (EnvA) into the same area and examined brains 7 days later for EGFP⁺ signal. We detected the highest number of EGFP⁺ cells in the ARC (38%), probably representing the initially infected AgRP neurons, and possibly local afferents (Fig. 1b; Supplementary Fig. 2). We next evaluated distant upstream anatomical areas for EGFP⁺ neurons and found that the vast majority were located in two hypothalamic nuclei, the dorsal medial hypothalamus (DMH, 26%) which contains both glutamatergic and GABAergic neurons¹³ and the paraventricular hypothalamus (PVH, 18%) consisting primarily of glutamatergic neurons¹³ (Fig. 1b; Supplementary Fig. 2). Finally, we also observed a smaller number of EGFP⁺ cells in other hypothalamic sites (Supplementary Fig. 2).

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

We next used channelrhodopsin (ChR2)-assisted circuit mapping (CRACM)^14,15 to both confirm and determine valence of functional monosynaptic connectivity between afferents and AgRP neurons. CRACM involves in vivo targeted expression of ChR2, a photoexcitable cation channel, in presumptive presynaptic upstream neurons (and their terminals), followed by ex vivo electrophysiologic assessment in acute brain slices of light-evoked postsynaptic currents in candidate downstream neurons. To investigate excitatory input to AgRP neurons, we stereotaxically injected Cre-dependent AAV expressing ChR2–mCherry (AAV8-DIO-ChR2–mCherry) (Supplementary Fig. 3a) into brain sites of Vglut2-IRES-Cre; Npy-hrGFP mice¹³. VGLUT2 (also known as SLC17A6) is the glutamate synaptic vesicle transporter expressed in the hypothalamus, hence Vglut2-IRES-Cre mice target relevant excitatory neurons¹³. As AgRP neurons co-express neuropeptide Y (NPY), Npy-hrGFP mice allow visualization of AgRP neurons^16,17. Consistent with the rabies tracing, we detected light-evoked excitatory post-synaptic currents (EPSCs) in all VGLUT2^DMH→AgRP^ARC neurons tested (latency between onset of light and EPSC = 4.7 ± 0.2 ms; Fig. 1c; Supplementary Fig. 3f). These were blocked by CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), an AMPA receptor antagonist, confirming their glutamatergic nature. Next, we examined monosynaptic connections between VGLUT2^PVH→AgRP^ARC neurons and again, consistent with the rabies mapping, we observed light-evoked EPSCs in all AgRP neurons tested (latency = 4.9 ± 0.4 ms; Fig. 1d; Supplementary Fig. 3g). These also were blocked by CNQX.

In addition, we selectively expressed ChR2 in the ventral medial hypothalamus (VMH) and lateral hypothalamus (LH), two sites with few EGFP⁺ cells, and also the ARC, which could provide local afferents, and investigated possible connectivity to AgRP neurons. In agreement with the negative rabies data, no light-evoked EPSCs were detected in 36 out of 37 VGLUT2^VMH→AgRP^ARC neurons tested (Supplementary Fig. 3b, h) or in any VGLUT2^LH→AgRP^ARC neurons tested (Supplementary Fig. 3c, i). Likewise, we failed to detect light-evoked EPSCs in any VGLUT2^ARC→AgRP^ARC neurons tested (Supplementary Fig. 3d, j). However, and as previously noted¹⁸, glutamatergic VMH neurons were monosynaptically connected to nearby pro-opiomelanocortin (POMC) neurons (VGLUT2^VMH→POMC^ARC), as we observed light-evoked EPSCs in all POMC neurons tested (latency = 4.4 ± 0.2 ms; Supplementary Fig. 3e).

The CRACM studies suggest marked differences in the strength of VGLUT2^PVH→AgRP^ARC versus VGLUT2^DMH→AgRP^ARC inputs. First, the amplitude of light-evoked EPSCs generated from VGLUT2^PVH inputs were approximately threefold greater (Fig. 1e). Second, the effectiveness of light pulses in evoking EPSCs differed, with DMH inputs showing a much higher failure rate (∼32% ; Fig. 1f; Supplementary Fig. 4) compared with PVH inputs in which time-locked EPSCs followed every light pulse (0% failure rate; Fig. 1g; Supplementary Fig. 4). To test for consequences of these synaptic strength differences, we assessed the ability of VGLUT2^DMH→AgRP^ARC or VGLUT2^PVH→AgRP^ARC inputs to fire action potentials in AgRP neurons. Whereas we never detected VGLUT2^DMH→AgRP^ARC light-evoked action potentials (Fig. 1h), we observed VGLUT2^PVH→AgRP^ARC light-evoked action potentials approximately 75% of the time (Fig. 1i), demonstrating potent, excitatory input to AgRP neurons originating from the PVH.

To determine which PVH neurons monosynaptically drive AgRP neurons, we used many lines of Cre-expressing mice, each targeting subsets of PVH neurons, and then used CRACM to determine connectivity. Sim1 encodes for the single-minded homologue 1 transcription factor required for developmental specification of the PVH, and is thus expressed in most PVH neurons¹⁹, as verified in the Sim1-Cre transgenic mouse²⁰. We observed light-evoked EPSCs in all SIM1^PVH→AgRP^ARC neurons tested (latency = 4.6 ± 0.4 ms; Fig. 2a). We next surveyed the Allen Brain Atlas for mRNAs highly enriched in the PVH, and then assembled a series of IRES-Cre knock-in mice targeting cells marked by prodynorphin (Pdyn-IRES-Cre; Supplementary Fig. 5), oxytocin (Oxt-IRES-Cre)²¹, arginine vasopressin (Avp-IRES-Cre; Supplementary Fig. 6), corticotropin-releasing hormone (Crh-IRES-Cre; Supplementary Fig. 7), TRH (Trh-IRES-Cre; Supplementary Fig. 8) and PACAP (Pacap-IRES-Cre; Supplementary Fig. 9). Following ChR2 delivery into the PVH, we failed to detect light-evoked EPSCs in all PDYN^PVH→AgRP^ARC (Fig. 2b), OXT^PVH→AgRP^ARC (Supplementary Fig. 10a), AVP^PVH→AgRP^ARC (Supplementary Fig. 10b) or CRH^PVH→AgRP^ARC (Supplementary Fig. 10c) neurons tested. However, we detected light-evoked EPSCs in all TRH^PVH→AgRP^ARC neurons (latency = 4.7 ± 0.4 ms; Fig. 2c) and all PACAP^PVH→AgRP^ARC neurons tested (latency = 4.8 ± 0.3 ms; Fig. 2d). These findings demonstrate that TRH^PVH and PACAP^PVH neurons provide excitatory drive to AgRP neurons. It is not known if these excitatory inputs come from two distinct classes of PVH neurons or from one which co-expresses TRH and PACAP. The latter is possible given that 37% of TRH mRNA-expressing neurons are marked by Pacap-IRES-Cre (Supplementary Fig. 11).

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

Innervation by PACAP^PVH neurons suggests that PACAP, in addition to glutamate, could also activate AgRP neurons. To assess this, we exogenously added PACAP_1–38 (100 nM)²² and determined effects on activity of synaptically isolated AgRP neurons (in the presence of picrotoxin (PTX) and kynurenate). Of note, PACAP_1–38 markedly depolarized and increased firing rate, and this was prevented by co-addition of the PAC1-receptor blocker, PACAP_6–38 (200 nM) (Fig. 2e–h). Thus, in addition to glutamate, PACAP working through PAC1 receptors also probably plays an important role in the PACAP^PVH→AgRP^ARC circuit.

POMC and AgRP neurons lie in close proximity within the ARC, but have opposing effects on feeding^2,23. If fidelity of the identified PVH→AgRP^ARC neuron hunger circuit is to be high, then it should not also engage satiety-promoting POMC neurons. We evaluated this and found that the vast majority of TRH^PVH→POMC^ARC neurons (21 of 22) and all PACAP^PVH→POMC^ARC neurons recorded are not monosynaptically connected (Fig. 3a, b, respectively). However, consistent with our observation that VGLUT2^VMH→POMC^ARC neurons are connected (Supplementary Fig. 3e), and as a positive control for studies investigating PACAP→POMC^ARC connections, we detected PACAP^VMH→POMC^ARC monosynaptic connections (latency = 4.5 ± 0.2 ms; Fig. 3c). These studies clearly demonstrate that the identified PVH→AgRP^ARC neuron circuit selectively drives hunger-promoting AgRP neurons, but not satiety-promoting POMC neurons.

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

Fidelity should also exist in the downstream connections of orexigenic AgRP neurons, which have reciprocal connections with the PVH. GABAergic AgRP neurons are monosynaptically connected with a subset of satiety-promoting neurons in the PVH, and this inhibitory connection drives feeding⁵. Here we confirm this inhibitory AgRP^ARC→PVH connection following ChR2 expression in AgRP neurons, as we detected light-evoked IPSCs (latency = 5.2 ± 0.3 ms) in a subset (55%) of AGRP^ARC→SIM1^PVH neurons (Fig. 3d), which were blocked by PTX. If fidelity of this hunger-promoting, GABAergic reciprocal AgRP^ARC→PVH connection is to be high, then it should not also engage and consequently inhibit the TRH^PVH and PACAP^PVH neurons. As expected, we detected no AgRP^ARC→TRH^PVH connections (Fig. 3e) and only rare AgRP^ARC→PACAP^PVH connections (1 of 12 neurons tested) (Fig. 3f). The above studies demonstrate marked fidelity in the reciprocal TRH/PACAP^PVH glutamatergic to AgRP^ARC GABAergic to satiety^PVH neuron circuit that drives feeding (Fig. 3g).

To confirm function of the TRH^PVH and PACAP^PVH neurons, we targeted DREADDs (designer receptors exclusively activated by designer drugs; AAV8-DIO-hM3Dq-mCherry)³ to the PVH. Upon binding of clozapine-N-oxide (CNO), hM3Dq activates neurons through the Gq signalling cascade²⁴. After verifying in brain slices that CNO depolarized and increased firing frequency of TRH^PVH and PACAP^PVH neurons (Fig. 4a, d, respectively), we found that acutely stimulating upstream TRH^PVH or PACAP^PVH neurons markedly induced Fos activity in AgRP cells, ipsilateral to the DREADD-activated upstream PVH neurons (Fig. 4b, e, respectively).

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

We next assessed effects of bilateral h3MDq-DREADD-mediated TRH^PVH or PACAP^PVH neuron stimulation on feeding behaviour during the light cycle, a time when food intake is usually low because of feeding during the preceding dark cycle (as observed following saline injections, black lines in Fig. 4c, f, respectively). Importantly, acute activation by CNO injection of either TRH^PVH or PACAP^PVH neurons caused robust feeding (red lines in Fig. 4c, f, respectively). This effect was absent following an overnight fast which itself elevates excitatory drive and consequently AgRP neuron activity^1,8,9 (Supplementary Fig. 12). To directly demonstrate that this enhanced feeding was mediated through AgRP neurons, we activated TRH^PVH neurons (AAV8-DIO-hM3Dq-mCherry) while simultaneously inhibiting AgRP^ARC neurons (AAV8-DIO-hM4Di-mCherry³; Fig. 4g). This significantly attenuated feeding (Fig. 4h). Finally, to examine whether endogenous activity of the TRH^PVH→AgRP^ARC pathway is physiologically relevant for feeding, we bilaterally transduced TRH^PVH neurons with inhibitory DREADDs, which led to rapid hyperpolarization and decreased firing rate upon CNO application (as assessed in slice studies, Fig. 4i), and found that this manipulation drastically reduced food intake during the dark cycle (Fig. 4j). These studies demonstrate both the sufficiency and necessity of this PVH→AgRP^ARC orexigenic circuit in regulating food intake.

Given the vast number of studies implicating an anorexigenic role for the PVH^{5,20,25,26,27,28}, it is remarkable to now discover embedded with the PVH a subset of neurons that drive feeding, through a reciprocal circuit (Fig. 3g). Failure of prior studies to detect orexigenic activity in the PVH is probably due to the reciprocal nature of this hunger circuit (leaving and returning to the PVH) and the inhibitory aspect of its return arm (AgRP^ARC→satiety^PVH neurons).

Methods Summary

All experiments were conducted according to US National Institutes of Health guidelines for animal research and were approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee.

Mice

Pdyn-IRES Cre, Avp-IRES-Cre, Crh-IRES-Cre, Trh-IRES-Cre and Pacap-IRES-Cre and R26-loxSTOPlox-L10-GFP mice were generated using recombineering techniques as previously described^13,20.

Viral injections

Stereotaxic injections were performed as previously described^3,4,29.

Rabies cell counting

For each brain, images were taken throughout one entire brain series, EGFP⁺ cells were quantified and assigned to a specific anatomical structure, and subsequently expressed as a percentage of the total number of cells counted for each mouse, averaged over the entire cohort.

Electrophysiology

Electrophysiology experiments were performed as previously described²⁹. Latencies between onset of light and post synaptic currents are expressed as mean ± s.e.m.

In situ hybridization

Digoxigenin-labelled riboprobes against Pacap, Pdyn or Trh were hybridized to brain sections of Pacap-IRES-Cre; R26-loxSTOPlox-L10-GFP, Pdyn-IRES-Cre; R26-loxSTOPlox-L10-GFP or Trh-IRES-Cre mice injected bilaterally with AAV-DIO-GFP into the PVH as previously described with minor modifications³⁰. Signals from intrinsic Cre-mediated GFP expression and in situ hybridization were compared to determine the degree of co-localization.

Fos assay

Assessment of Fos induction used a previously developed method⁸ modified for fluorescent co-localization with hrGFP in AgRP neurons.

Food intake

Food intake studies on chow were performed as previously described^3,4.

Statistics

Statistical analyses were performed using Origin Pro 8.6 and Prism 6.0 (GraphPad) software. Feeding studies were run as within-subject design and a final consumption value for each animal obtained from an average of 3 trials. Data was analysed using a two-way repeated-measures ANOVA, interaction of ‘time’ and ‘treatment’ or ‘genotype’ and ‘treatment’. *P < 0.05, **P < 0.001. Error bars indicate mean +/− s.e.m. For slice electrophysiology experiments and cell counting analyses, P values for pair-wise or unpaired comparisons were calculated by two-tailed Student’s t-test. *P < 0.05, **P < 0.001.

Online Content Any additional Methods, Extended Data display items and Source Data are available in the online version of the paper; references unique to these sections appear only in the online paper.

Online Methods

Animals

All animal care and experimental procedures were approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee. Mice were housed at 22–24 °C with a 12 h light:12 h dark cycle with standard mouse chow (Teklad F6 Rodent Diet 8664; 4.05 kcal g⁻¹, 3.3 kcal g⁻¹ metabolizable energy, 12.5% kcal from fat; Harlan Teklad) and water provided ad libitum. All diets were provided as pellets. Mice were euthanized by CO₂ narcosis.

Generation of mice

Pdyn-IRES Cre, Avp-IRES-Cre, Crh-IRES-Cre, Trh-IRES-Cre and Pacap-IRES-Cre mice were generated using recombineering techniques as previously described^13,21. Briefly, a selection cassette containing an internal ribosomal entry sequence (IRES) linked to Cre- recombinase and an Frt-flanked kanamycin resistance gene was targeted just downstream of the stop codon of the prodynorphin, arginine vasopressin, corticotropin releasing hormone, thyrotropin releasing hormone or adenylate cyclase activating peptide 1 gene, respectively, in a bacterial artificial chromosome, so that Cre recombinase expression was driven by the endogenous genes. A targeting plasmid containing the Cre-containing selection cassette and 4 kb genomic sequence upstream and downstream of the prodynorphin, arginine vasopressin, corticotropin releasing hormone, thyrotropin releasing hormone or adenylate cyclase activating peptide 1 stop codon, respectively was isolated and used for embryonic stem cell targeting. Correctly targeted clones were identified by long range PCR and injected into blastocysts. Chimaeric animals generated from blastocyst implantation were then bred for germline transmission of the altered prodynorphin, arginine vasopressin, corticotropin releasing hormone, thyrotropin releasing hormone or adenylate cyclase activating peptide 1-allele, respectively. Flp-deletion mice were then used to remove the neomycin selection cassette.

Generation of an enhanced Cre-dependent GFP reporter mice (R26-loxSTOPlox-L10-GFP) were generated using recombineering techniques as previously described²⁰. A transgene containing a lox-flanked transcriptional blocking cassette followed by EGFP fused to the L10-ribosomal subunit³¹ was placed under the control of a CMV-enhancer/chicken β-actin promoter and targeted to the Rosa26 locus using standard techniques³². Correctly targeted blastocysts were identified by long range PCR and confirmed by Southern blotting and injected into blastocysts.

Characterization of mice

Pdyn-IRES-Cre, Avp-IRES-Cre, Crh-IRES-Cre, and Pacap-IRES-Cre mice were crossed to R26-loxSTOPlox-L10-GFP mice, euthanized and sectioned at 30 μm. Brain sections were washed in 0.1 M phosphate-buffered saline with Tween 20, pH 7.4 (PBST, 2 changes) and then incubated in the primary antiserum (anti-GFP, Abcam (1:1,000), rabbit anti-hrGFP. After several washes in PBS, sections were incubated in Alexa fluorophore secondary antibody (Molecular Probes, 1:200) for 2 h at room temperature. After several washes in PBS, sections were mounted onto gelatin-coated slides and fluorescent images were captured with Olympus VS120 slide scanner microscope.

Trh-IRES-Cre mice were stereotaxically injected with Cre-dependent AAV8-DIO-mCherry⁸, due to transient, early embryonic expression and subsequent deletion of floxed alleles by this mouse line, euthanized and sectioned at 30 μm. Native fluorescence was used and fluorescent images were captured with Olympus VS120 slide scanner microscope. All digital images were processed in the same way between experimental conditions to avoid artificial manipulation between different data sets.

In situ mRNA hybridization experiments were performed for Pdyn-IRES-Cre, Trh-IRES-Cre and Pacap-IRES-Cre mice (details below). Antibody staining experiments were performed for Avp-IRES-Cre and Crh-IRES-Cre mice. Briefly, Avp-IRES-Cre; R26-loxSTOPlox-L10-GFP mice were co-localized with anti-AVP, Sigma (1:500), rabbit anti-AVP and anti-GFP, Abcam (1:1,000), chicken anti-GFP. After 48 h intracerebroventricular colchicine treatment (Sigma, 10 μg), Crh-IRES-Cre; R26-loxSTOPlox-L10-GFP mice were co-localized with anti-CRF, Peninsula Laboratories (1:2,500), rabbit anti-CRF and anti-GFP, Abcam (1:1,000), chicken anti-GFP.

All images were subsequently compared to in situ mRNA expression profiles generated by the Allen Institute for Brain Science; Allen Mouse Brain Atlas 2013 (http://mouse.brain-map.org/).

Breeding strategies

Vglut2-IRES-Cre, Sim1-Cre, Pdyn-IRES-Cre, Oxt-IRES-Cre, Avp-IRES-Cre, Crh-IRES-Cre, Trh-IRES-Cre and Pacap-IRES-Cre mice were bred to Npy-hrGFP transgenic mice to generate heterozygous Vglut2-IRES-Cre; Npy-hrGFP, Sim1-Cre; Npy-hrGFP, Pdyn-IRES-Cre; Npy-hrGFP, Oxt-IRES-Cre; Npy-hrGFP, Avp-IRES-Cre; Npy-hrGFP, Crh-IRES-Cre; Npy-hrGFP, Trh-IRES-Cre; Npy-hrGFP and Pacap-IRES-Cre; Npy-hrGFP mice, respectively as previously reported⁸. Vglut2-IRES-Cre and Pacap-IRES-Cre mice were bred to Pomc-hrGFP transgenic mice to generate heterozygous Vglut2-IRES-Cre; Pomc-hrGFP and Pacap-IRES-Cre; Pomc-hrGFP mice, respectively as previously reported¹³.

Agrp-IRES-Cre, Pdyn-IRES-Cre, Avp-IRES-Cre, Crh-IRES-Cre and Pacap-IRES-Cre mice were bred to R26-loxSTOPlox-L10-GFP mice to generate heterozygous Agrp-IRES-Cre; R26-loxSTOPlox-L10-GFP, Pdyn-IRES-Cre; R26-loxSTOPlox-L10-GFP, Avp-IRES-Cre; R26-loxSTOPlox-L10-GFP, Crh-IRES-Cre; R26-loxSTOPlox-L10-GFP and Pacap-IRES-Cre; R26-loxSTOPlox-L10-GFP mice, respectively. Agrp-IRES-Cre; R26-loxSTOPlox-L10-GFP mice were then bred to Sim1-Cre and Pacap-IRES-Cre to generate heterozygous Agrp-IRES-Cre; Sim1-Cre R26-loxSTOPlox-L10-GFP and Agrp-IRES-Cre; Pacap-IRES-Cre; R26-loxSTOPlox-L10-GFP mice, respectively. All mice are on a mixed background.

Viral injections

Stereotaxic injections were performed as previously described³. Mice were anaesthetized with xylazine (5 mg per kg) and ketamine (75 mg per kg) diluted in saline (350 mg per kg) and placed into a stereotaxic apparatus (KOPF Model 963). After exposing the skull via small incision, a small hole was drilled for injection. A pulled-glass pipette with 20–40 mm tip diameter was inserted into the brain and virus was injected by an air pressure system. A micromanipulator (Grass Technologies, Model S48 Stimulator) was used to control injection speed at 25 nl min⁻¹ and the pipette was withdrawn 5 min after injection. For retrograde rabies tracing, AAV-FLEX-TVA-mCherry, serotype 8 (titre 1.1 × 10¹² genomes copies per ml) and AAV-FLEX-RG, serotype 8 (titre 1.4 × 10¹² genomes copies per ml)¹² were injected unilaterally into the ARC (200 nl, coordinates, bregma: AP:−1.50 mm, DV:−5.80 mm, L: −0.20 mm) of 5–6-weeks-old mice. Then 21 days later SADΔG–EGFP (EnvA) rabies (titre 10⁷genomes copies per ml) was unilaterally injected into the ARC (400 nl, coordinates, bregma: AP:−1.50 mm, DV:−5.80 mm, L: −0.20 mm). For electrophysiology experiments, AAV-DIO-ChR2(H134R)-mCherry, serotype 8 (titre 1.3 × 10¹² genomes copies per ml)²⁹ was injected unilaterally into the DMH (20 nl, coordinates, bregma: AP: −1.70 mm, DV: −5.00 mm, L: −0.25 mm), PVH (25 nl, coordinates, bregma: AP: −0.70 mm, DV: −4.75 mm, L: −0.20 mm), VMH (25 nl, coordinates, bregma: AP: −1.40 mm, DV: −5.60 mm, L: −0.40 mm) or ARC (20 nl, coordinates, bregma: AP: –1.50 mm, DV: −5.80 mm, L: −0.20 mm) of 5–6-weeks-old mice. For feeding and Fos studies, AAV-DIO-hM3Dq-mCherry, serotype 8 (titre 1.2 × 10¹² genomes copies per ml)³ or AAV-DIO-hM4Di-mCherry, serotype 8 (titre 1.7 × 10¹² genomes copies per ml)³ was injected bilaterally or unilaterally, respectively into the PVH (25 nl, coordinates, bregma: AP: –0.70 mm, DV: −4.75 mm, L: −0.20 mm) of 5–6-weeks-old mice. For validation of Trh-IRES-Cre mice, AAV-DIO-mCherry, serotype 8 (titre 1.2 × 10¹² genomes copies per ml)⁸ or AAV-DIO-GFP, serotype 8 (titre 1.4 × 10¹² genomes copies per ml) was injected bilaterally into the PVH (25 nl, coordinates, bregma: AP: −0.70 mm, DV: −4.75 mm, L: −0.20 mm) of 5−6-weeks-old mice. All viruses were packaged at the Gene Therapy Center at the University of North Carolina. For postoperative care, mice were injected intraperitoneally with meloxicam (0.5 mg per kg). All stereotaxic injection sites were verified under electrophysiological microscopy (for electrophysiology-related studies) or by immunohistochemistry (for anatomy and in vivo studies). All ‘misses’ or ‘partial hits’ were excluded from data analyses.

SADΔG–EGFP (EnvA) rabies cell counting

One week after SADΔG–EGFP (EnvA) rabies injection, mice were perfused and brains were sectioned and mounted as described above. For each brain (n = 6), ×10 magnification images were taken throughout one entire brain series using a Zeiss AxioImager Z.1 microscope and EGFP⁺ cells were quantified using these images. Each EGFP⁺ cell was assigned to a specific anatomical structure of the hypothalamus using The Mouse Brain in Stereotaxic Coordinates (Franklin & Paxinos). As the number of labelled cell varied over multiple animals depending on the transduction rate of the viruses, the counted cells in each anatomical structure were expressed as a percentage of the total number of cells counted throughout each mouse brain. The percentages for each anatomical structure in a given mouse were then averaged over the entire cohort of mice (Supplementary Fig. 2).

Electrophysiology and circuit mapping

For brain slice preparation, 6–10-weeks-old mice were anaesthetized with isoflurane before decapitation and removal of the entire brain. Brains were immediately submerged in ice-cold, carbogen-saturated (95% O₂, 5% CO₂) high sucrose solution (238 mM sucrose, 26 mM NaHCO₃, 2.5 mM KCl, 1.0 mM NaH₂PO₄, 5.0 mM MgCl₂, 10.0 mM CaCl₂, 11 mM glucose). Then, 300-µM thick coronal sections were cut with a Leica VT1000S Vibratome and incubated in oxygenated aCSF (126 mM NaCl, 21.4 mM NaHCO₃, 2.5 mM KCl, 1.2 mM NaH₂PO₄, 1.2 mM MgCl₂, 2.4 mM CaCl₂, 10 mM glucose) at 34 °C for 30 min. Then, slices were maintained and recorded at room temperature (20–24 °C). The intracellular solution for voltage clamp recording contained the following (in mM): 140 CsCl, 1 BAPTA, 10 HEPES, 5 MgCl₂, 5 Mg-ATP, 0.3 Na₂GTP, and 10 lidocaine N-ethyl bromide (QX-314), pH 7.35 and 290 mOsm. The intracellular solution for current clamp recordings contained the following (in mM): 128 K gluconate, 10 KCl, 10 HEPES, 1 EGTA, 1 MgCl₂, 0.3 CaCl₂, 5 Na₂ATP, 0.3 NaGTP, adjusted to pH 7.3 with KOH.

Photostimulation-evoked EPSCs and IPSCs were recorded in the whole cell voltage clamp mode, with membrane potential clamped at −60 mV. Photostimulation-evoked EPSCs was recorded in presence of picrotoxin (100 µM) to block inhibitory postsynaptic currents. All recordings were made using multiclamp 700B amplifier, and data were filtered at 2 kHz and digitized at 10 kHz. To photostimulate channelrhodopsin2-positive fibres, a laser or LED light source (473 nm; Opto Engine; Thorlabs) was used. The blue light was focused on to the back aperture of the microscope objective, producing a wide-field exposure around the recorded cell of 10–15 mW mm². The light power at the specimen was measured using an optical power meter PM100D (Thorlabs). The light output is controlled by a programmable pulse stimulator, Master-8 (A.M.P.I.) and the pClamp 10.2 software (Axon Instruments). Photostimulation-evoked EPSCs/IPSCs detection protocol constitutes four blue light laser pulses administered 1 s apart during the first 4 s of a 8 s sweep, repeating for a total of 30 sweeps. When recording photostimulation-evoked action potentials in AgRP neurons, current was injected into cells to keep the base line membrane potential at approximately −60 mV. Mice with total misses, partial expression or expression outside the intended area were excluded from analysis after examination of mCherry expression. VGLUT2^DMH→AgRP^ARC n = 4; VGLUT2^PVHH→AgRP^ARC n = 5; VGLUT2^VMH→AgRP^ARC n = 4; VGLUT2^LH→AgRP^ARC n = 3; VGLUT2^ARC→AgRP^ARC n = 3; VGLUT2^VMH→POMC^ARC n = 3; SIM1^PVH→AgRP^ARC n = 3; PDYN^PVH→AgRP^ARC n = 3; OXT^PVH→AgRP^ARC n = 3; AVP^PVH→AgRP^ARC n = 3; CRH^PVH→AgRP^ARC n = 3; TRH^PVH→AgRP^ARC n = 3; PACAP^PVH→AgRP^ARC n = 4; TRH^PVH→POMC^ARC n = 4; PACAP^PVH→POMC^ARC n = 3; PACAP^VMH→POMC^ARC n = 2; AgRP^ARC→SIM1^PVH n = 2; AgRP^ARC→TRH^PVH n = 3; AgRP^ARC→PACAP^PVH n = 3.

To assess the effect of PACAP_1–38 (100 nM)²² and PACAP_6–38 (200 nM) onto AgRP neurons, we performed whole cell current clamp recordings on to 5–8-weeks-old Npy-hrGFP mice. Synaptic blockers (1 mM kynurenate and 100 µM picrotoxin) were added in aCSF to synaptically isolate AgRP neurons.

To assess the effect of CNO on to TRH and PACAP neurons, 5- to 7-week-old Pacap-IRES-Cre and Trh-IRES-Cre mice were injected with AAV8-DIO-hM3Dq-mCherry into the PVH 2–3 weeks before recording. CNO was applied to bath solution through perfusion as previously reported³. After acquisition of stable whole-cell recordings for 2–5 min, aCSF solution containing 5 µM CNO was perfused into the brain slice preparation.

Fos analysis

Animals (Trh-IRES-Cre; Npy-hrGFP; n = 3 and Pacap-IRES-Cre; Npy-hrGFP; n = 3 mice), were handled for 10 consecutive days before the assay to reduce stress response, and then injected with CNO (0.3 mg per kg; i.p.) at 9:00. Then 150 min later, the animals were euthanized with 7% chloral hydrate diluted in saline (350 mg per kg) for histological assay. The mice were perfused and brains were sectioned as described above. Assessment of Fos induction was performed using a previously developed method⁸ modified for fluorescent co-localization with hrGFP in AgRP neurons. Brain sections were processed for immunohistochemical detection of Fos and hrGFP and counting.

Brain sections were washed in 0.1 M phosphate-buffered saline with Tween 20, pH 7.4 (PBST, 2 changes) and then incubated in the primary antiserum (anti-GFP, Abcam (1:1,000), rabbit anti-hrGFP and anti-Fos, Calbiochem (1:10,000). After several washes in PBS, sections were incubated with Alexa fluorophore secondary antibodies (Molecular Probes, 1:200) for 2 h at room temperature. After several washes in PBS, sections were mounted onto gelatin-coated slides and fluorescent ×10 magnification images were taken initially using a Zeiss AxioImager Z.1 microscope. Co-localization and quantification was further determined using fluorescent ×20 magnification images taken using a Zeiss AxioImager Z.1 microscope. Data are expressed as the percentage of all AgRP neurons (that is, all hrGFP-positive neurons) that were double-positive for Fos and hrGFP.

Food intake studies

Food intake studies on chow were performed as previously described^3,8,29. All animals (10- to 12-week-old male mice) were singly housed for at least 2.5 weeks following surgery and handled for 10 consecutive days before the assay to reduce stress response. Feeding studies were performed in home cages with ad libitum food access. CNO was administered at 0.3 mg per kg of body weight. Saline was delivered at the same volume to maintain consistency in the studies. Previous publications suggest the duration of the drugs effect at the dosage used throughout the studies is approximately 8 h²⁴. Mice with ‘missed’ injections, incomplete ‘hits’ or expression outside the area of interest were excluded from analysis after post hoc examination of mCherry expression. In this way, all food intake measurements were randomized and blind to the experimenter.

For light cycle measurements, animals (Trh-IRES-Cre; n = 8, Pacap-IRES-Cre; n = 7 mice; Fig. 4c, f, or Trh-IRES-Cre; Agrp-IRES-Cre; n = 4, Trh-IRES-Cre; n = 4 mice; Fig. 4h) were injected with either saline or CNO (0.3 mg per kg; i.p.) at 9:00 and food intake was monitored 1 h and/or 2 h after i.p. injection from 9:00 to 11:00. A full trial consisted of assessing food intake from the study subjects after they received injections of saline on day 1 and CNO on day 2. Animals received a day ‘off’ between trials before another trial was initiated. The food intake data from all days following saline/CNO (n = 24 trials for Trh-IRES-Cre; n = 21 trials for Pacap-IRES-Cre; Fig. 4c, f; n = 12 for Trh-IRES-Cre; Agrp-IRES-Cre; n = 12 trials for Trh-ires-Cre; Fig. 4h) injections were then averaged and combined for analysis.

For dark cycle measurements, animals (Trh-IRES-Cre; n = 4 mice; Fig. 4j) were injected with either saline or CNO (0.3 mg per kg; i.p.) at 18:00 and food intake was monitored 1, 2 and 3 h after i.p. injection from 18:00 to 21:00. A full trial consisted of assessing food intake from the study subjects after they received injections of saline on day 1 and CNO on day 2. Animals received a day ‘off’ between trials before another trial was initiated. The food intake data from all days following saline/CNO (n = 12 trials for Trh-IRES-Cre; Fig. 4j) injections was then averaged and combined for analysis.

For fast-refeed measurements, animals (Trh-IRES-Cre; n = 8, Pacap-IRES-Cre; n = 7 mice; Supplementary Fig. 12) were fasted overnight at 17:00 and the following day were injected with either saline or CNO (0.3 mg per kg; i.p.) at 8:30 and food intake was monitored 1.5, 2.5 and 4.5 h after i.p. injection from 9:00 to 13:00. A full trial consisted of assessing food intake from the study subjects after they received injections of saline on week 1 and CNO on week 2. Animals received a week ‘off’ between trials before another trial was initiated. The food intake data from all days following saline/CNO (n = 16 trials for Trh-IRES-Cre; n = 14 trials for Pacap-IRES-Cre; Supplementary Fig. 12) injections were then averaged and combined for analysis.

In situ hybridization

Pacap-IRES-Cre; R26-loxSTOPlox-L10-GFP and Pdyn-IRES-Cre; R26-loxSTOPlox-L10-GFP mice were euthanized and brains were frozen fresh. Trh-IRES-Cre mice with bilateral AAV-DIO-GFP injections into the PVH were perfused with 4% PFA, post-fixed for 2 h and equilibrated in 20% sucrose overnight. Then 14-μM cryosections were analysed by in situ hybridization as previously described³⁰ with minor modifications. Slides were fixed (4% PFA), washed (2× RNase-free PBS), coverslips were added and samples imaged for intrinsic Cre-mediated GFP fluorescence. Digoxigenin labelled anti-sense cRNA probes were generated by T3 (Roche) in vitro transcription reactions using the complete coding sequence of Pdyn (5′ primer: 5′-ATGGCGTGGTCCAGGCTGATGC-3′; 3′ primer: 5′-TCAAACATCTAAATCTTCAGAATAGG-3′) and a 914-bp fragment of Pacap cDNA (5′ primer: 5′-CTGCGTGACGCTTACGCCCT-3′; 3′ primer: 5′-TTGCCCCTGCAACCAGTGGG-3′). A previously described murine cDNA³³ (gift of Masanobu Yamada) was used to generate a Trh probe with SP6 RNA polymerase (Roche). Following hybridization, slides were incubated with anti-digoxigenin antibody conjugated to alkaline phosphatase (Roche, 1:200, 1 h room temperature), washed and incubated (3–6 h) in NBT/BCIP chromogenic substrate according to the manufacturer’s specifications (Roche). Slides had coverslips added and brightfield images were captured. Images were pseudocoloured and compared with intrinsic Cre-mediated GFP fluorescence for co-localization and quantification.

Statistical analysis

Statistical analyses were performed using Origin Pro 8.6 and Prism 6.0 (GraphPad) software.

References

Takahashi, K. A. & Cone, R. D. Fasting induces a large, leptin-dependent increase in the intrinsic action potential frequency of orexigenic arcuate nucleus neuropeptide Y/Agouti-related protein neurons. Endocrinology 146, 1043–1047 (2005)
Article CAS Google Scholar
Aponte, Y., Atasoy, D. & Sternson, S. M. AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training. Nature Neurosci. 14, 351–355 (2011)
Article CAS Google Scholar
Krashes, M. J. et al. Rapid, reversible activation of AgRP neurons drives feeding behavior in mice. J. Clin. Invest. 121, 1424–1428 (2011)
Article CAS Google Scholar
Krashes, M. J., Shah, B. P., Koda, S. & Lowell, B. B. Rapid versus delayed stimulation of feeding by the endogenously released AgRP neuron mediators GABA, NPY, and AgRP. Cell Metab. 18, 588–595 (2013)
Article CAS Google Scholar
Atasoy, D., Betley, J. N., Su, H. H. & Sternson, S. M. Deconstruction of a neural circuit for hunger. Nature 488, 172–177 (2012)
Article ADS CAS Google Scholar
Luquet, S., Perez, F. A., Hnasko, T. S. & Palmiter, R. D. NPY/AgRP neurons are essential for feeding in adult mice but can be ablated in neonates. Science 310, 683–685 (2005)
Article ADS CAS Google Scholar
Gropp, E. et al. Agouti-related peptide-expressing neurons are mandatory for feeding. Nature Neurosci. 8, 1289–1291 (2005)
Article CAS Google Scholar
Liu, T. et al. Fasting activation of AgRP neurons requires NMDA receptors and involves spinogenesis and increased excitatory tone. Neuron 73, 511–522 (2012)
Article Google Scholar
Yang, Y., Atasoy, D., Su, H. H. & Sternson, S. M. Hunger states switch a flip-flop memory circuit via a synaptic AMPK-dependent positive feedback loop. Cell 146, 992–1003 (2011)
Article CAS Google Scholar
Pinto, S. et al. Rapid rewiring of arcuate nucleus feeding circuits by leptin. Science 304, 110–115 (2004)
Article ADS CAS Google Scholar
Wickersham, I. R., Finke, S., Conzelmann, K. K. & Callaway, E. M. Retrograde neuronal tracing with a deletion-mutant rabies virus. Nature Methods 4, 47–49 (2007)
Article CAS Google Scholar
Watabe-Uchida, M., Zhu, L., Ogawa, S. K., Vamanrao, A. & Uchida, N. Whole-brain mapping of direct inputs to midbrain dopamine neurons. Neuron 74, 858–873 (2012)
Article CAS Google Scholar
Vong, L. et al. Leptin action on GABAergic neurons prevents obesity and reduces inhibitory tone to POMC neurons. Neuron 71, 142–154 (2011)
Article CAS Google Scholar
Petreanu, L., Huber, D., Sobczyk, A. & Svoboda, K. Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections. Nature Neurosci. 10, 663–668 (2007)
Article CAS Google Scholar
Atasoy, D., Aponte, Y., Su, H. H. & Sternson, S. M. A. FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping. J. Neurosci. 28, 7025–7030 (2008)
Article CAS Google Scholar
Hahn, T. M., Breininger, J. F., Baskin, D. G. & Schwartz, M. W. Coexpression of Agrp and NPY in fasting-activated hypothalamic neurons. Nature Neurosci. 1, 271–272 (1998)
Article CAS Google Scholar
van den Pol, A. N. et al. Neuromedin B and gastrin-releasing peptide excite arcuate nucleus neuropeptide Y neurons in a novel transgenic mouse expressing strong Renilla green fluorescent protein in NPY neurons. J. Neurosci. 29, 4622–4639 (2009)
Article CAS Google Scholar
Sternson, S. M., Shepherd, G. M. & Friedman, J. M. Topographic mapping of VMH → arcuate nucleus microcircuits and their reorganization by fasting. Nature Neurosci. 8, 1356–1363 (2005)
Article CAS Google Scholar
Michaud, J. L., Rosenquist, T., May, N. R. & Fan, C. M. Development of neuroendocrine lineages requires the bHLH-PAS transcription factor SIM1. Genes Dev. 12, 3264–3275 (1998)
Article CAS Google Scholar
Balthasar, N. et al. Divergence of melanocortin pathways in the control of food intake and energy expenditure. Cell 123, 493–505 (2005)
Article CAS Google Scholar
Wu, Z. et al. An obligate role of oxytocin neurons in diet induced energy expenditure. PLoS ONE 7, e45167 (2012)
Article ADS CAS Google Scholar
Fukushima, Y. et al. Role of endogenous PACAP in catecholamine secretion from the rat adrenal gland. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R1562–R1567 (2001)
Article CAS Google Scholar
Zhan, C. et al. Acute and long-term suppression of feeding behavior by POMC neurons in the brainstem and hypothalamus, respectively. J. Neurosci. 33, 3624–3632 (2013)
Article CAS Google Scholar
Alexander, G. M. et al. Remote control of neuronal activity in transgenic mice expressing evolved G protein-coupled receptors. Neuron 63, 27–39 (2009)
Article CAS Google Scholar
Leibowitz, S. F., Hammer, N. J. & Chang, K. Hypothalamic paraventricular nucleus lesions produce overeating and obesity in the rat. Physiol. Behav. 27, 1031–1040 (1981)
Article CAS Google Scholar
Gold, R. M., Jones, A. P. & Sawchenko, P. E. Paraventricular area: critical focus of a longitudinal neurocircuitry mediating food intake. Physiol. Behav. 18, 1111–1119 (1977)
Article CAS Google Scholar
Stanley, B. G. & Leibowitz, S. F. Neuropeptide Y injected in the paraventricular hypothalamus: a powerful stimulant of feeding behavior. Proc. Natl Acad. Sci. USA 82, 3940–3943 (1985)
Article ADS CAS Google Scholar
Cowley, M. A. et al. Integration of NPY, AGRP, and melanocortin signals in the hypothalamic paraventricular nucleus: evidence of a cellular basis for the adipostat. Neuron 24, 155–163 (1999)
Article CAS Google Scholar
Kong, D. et al. GABAergic RIP-Cre neurons in the arcuate nucleus selectively regulate energy expenditure. Cell 151, 645–657 (2012)
Article CAS Google Scholar
Montmayeur, J. P., Liberles, S. D., Matsunami, H. & Buck, L. B. A candidate taste receptor gene near a sweet taste locus. Nature Neurosci. 4, 492–498 (2001)
Article CAS Google Scholar
Heiman, M. et al. A translational profiling approach for the molecular characterization of CNS cell types. Cell 135, 738–748 (2008)
Article CAS Google Scholar
Srinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4 (2001)
Article CAS Google Scholar
Satoh, T., Yamada, M., Monden, T., Iizuka, M. & Mori, M. Cloning of the mouse hypothalamic preprothyrotropin-releasing hormone (TRH) cDNA and tissue distribution of its mRNA. Brain Res. Mol. Brain Res. 14, 131–135 (1992)
Article CAS Google Scholar

Download references

Acknowledgements

This research was funded by the following NIH grants to B.B.L.: R01 DK096010, R01 DK089044, R01 DK071051, R01 DK075632, R37 DK053477, BNORC Transgenic Core P30 DK046200 and BADERC Transgenic Core P30 DK57521; to M.J.K.: F32 DK089710; to D.P.O.: K08 DK071561; to L.V.: F32 DK078478; to N.U.: R01 MH095953; and an ADA Mentor-Based Fellowship to B.P.S. and B.B.L. We thank D. Cusher and Y. Li for mouse genotyping, J. Carroll and Y. Guo for perfusions and tissue removal, B. L. Roth and S. C. Rogan for generating the AAV-DIO-hM3Dq-mCherry plasmid, K. Deisseroth for generating the AAV-DIO-ChR2-mCherry plasmid and A. N. Hollenberg for the TRH riboprobe.

Author information

Michael J. Krashes, Bhavik P. Shah, David P. Olson & Linh Vong
Present address: Present addresses: Diabetes, Endocrinology and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA (M.J.K.); National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland 21224, USA (M.J.K.); Cardiovascular and Metabolic Diseases, Pfizer, 610 Main Street, Cambridge, Massachusetts 02139, USA (B.P.S.); Division of Pediatric Endocrinology, Departments of Pediatrics, University of Michigan, Ann Arbor, Michigan 48105, USA (D.P.O.); Cardiovascular and Metabolic Diseases, Novartis Institutes for BioMedical Research, 100 Technology Square, Cambridge, Massachusetts 02139, USA (L.V.).,
Michael J. Krashes and Bhavik P. Shah: These authors contributed equally to this work.

Authors and Affiliations

Division of Endocrinology, Department of Medicine, Diabetes and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, 02215, Massachusetts, USA
Michael J. Krashes, Bhavik P. Shah, Joseph C. Madara, David P. Olson, Alastair S. Garfield, Linh Vong & Bradford B. Lowell
Department of Cell Biology, Harvard Medical School, Boston, 02115, Massachusetts, USA
David E. Strochlic & Stephen D. Liberles
Program in Neuroscience, Harvard Medical School, Boston, 02115, Massachusetts, USA
David E. Strochlic, Naoshige Uchida, Stephen D. Liberles & Bradford B. Lowell
Center for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, UK,
Alastair S. Garfield
Division of Pediatric Endocrinology, Departments of Pediatrics, University of Michigan, Ann Arbor, 48105, Michigan, USA
Hongjuan Pei
Department of Molecular and Cellular Biology, Center for Brain Science, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138, USA,
Mitsuko Watabe-Uchida & Naoshige Uchida

Authors

Michael J. Krashes
View author publications
You can also search for this author in PubMed Google Scholar
Bhavik P. Shah
View author publications
You can also search for this author in PubMed Google Scholar
Joseph C. Madara
View author publications
You can also search for this author in PubMed Google Scholar
David P. Olson
View author publications
You can also search for this author in PubMed Google Scholar
David E. Strochlic
View author publications
You can also search for this author in PubMed Google Scholar
Alastair S. Garfield
View author publications
You can also search for this author in PubMed Google Scholar
Linh Vong
View author publications
You can also search for this author in PubMed Google Scholar
Hongjuan Pei
View author publications
You can also search for this author in PubMed Google Scholar
Mitsuko Watabe-Uchida
View author publications
You can also search for this author in PubMed Google Scholar
Naoshige Uchida
View author publications
You can also search for this author in PubMed Google Scholar
Stephen D. Liberles
View author publications
You can also search for this author in PubMed Google Scholar
Bradford B. Lowell
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

M.J.K., B.P.S. and B.B.L. designed the experiments and analysed data. M.J.K., B.P.S., J.C.M., D.E.S., A.S.G., L.V. and H.P. performed experiments. M.J.K. generated Pdyn-IRES-Cre, Trh-IRES-Cre and Pacap-IRES-Cre mice, D.P.O. generated Oxt-IRES-Cre, Avp-IRES-Cre, Crh-IRES-Cre and R26-loxSTOPlox-L10-GFP mice and L.V. generated Vglut2-IRES-Cre mice. M.W.-U. and N.U. generated, provided and advised on use of AAV-FLEX-TVA-mCherry, AAV-FLEX-RG and SADΔG–EGFP (EnvA) viruses. S.D.L. advised on in situ hybridization experiments. M.J.K and B.B.L. wrote the manuscript with comments from all of the authors.

Corresponding author

Correspondence to Bradford B. Lowell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-12. (PDF 28905 kb)

PowerPoint slides

PowerPoint slide for Fig. 1

PowerPoint slide for Fig. 2

PowerPoint slide for Fig. 3

PowerPoint slide for Fig. 4

Rights and permissions

Reprints and permissions

About this article

Cite this article

Krashes, M., Shah, B., Madara, J. et al. An excitatory paraventricular nucleus to AgRP neuron circuit that drives hunger. Nature 507, 238–242 (2014). https://doi.org/10.1038/nature12956

Download citation

Received: 22 April 2013
Accepted: 16 December 2013
Published: 02 February 2014
Issue Date: 13 March 2014
DOI: https://doi.org/10.1038/nature12956

This article is cited by

Daily feeding entrains hunger-sensing neurons
- Zane B. Andrews
Nature Neuroscience (2024)
AgRP neurons encode circadian feeding time
- Nilufer Sayar-Atasoy
- Iltan Aklan
- Deniz Atasoy
Nature Neuroscience (2024)
An airway-to-brain sensory pathway mediates influenza-induced sickness
- Na-Ryum Bin
- Sara L. Prescott
- Stephen D. Liberles
Nature (2023)
Neural basis for fasting activation of the hypothalamic–pituitary–adrenal axis
- Amelia M. Douglass
- Jon M. Resch
- Bradford B. Lowell
Nature (2023)
Cannabis Sativa targets mediobasal hypothalamic neurons to stimulate appetite
- Emma C. Wheeler
- Pique Choi
- Jon F. Davis
Scientific Reports (2023)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Subjects

Abstract

Similar content being viewed by others

Main

Methods Summary

Mice

Viral injections

Rabies cell counting

Electrophysiology

In situ hybridization

Fos assay

Food intake

Statistics

Online Methods

Animals

Generation of mice

Characterization of mice

Breeding strategies

Viral injections

SADΔG–EGFP (EnvA) rabies cell counting

Electrophysiology and circuit mapping

Fos analysis

Food intake studies

In situ hybridization

Statistical analysis

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Competing interests

Supplementary information

PowerPoint slides

Rights and permissions

About this article

Cite this article

Share this article

This article is cited by

Comments

Search

Quick links