Experimental protocols were approved by the University of California, San Francisco IACUC following the National Institutes of Health guidelines for the Care and Use of Laboratory Animals. Animals were housed in 12-hr dark/light cycle with ad libitum access to food and water. Prior to experiments, animals were fasted for 16 hr as noted in the main text; they maintained ad libitum access to water. Agrp tm1(cre)Lowl (AgRP Cre , #012899) and Tg(Pomc1-cre)16Lowl (POMC Cre , #005965) animals have been previously described and have been backcrossed onto a C57BL/6 background. To generate leptin-deficient AgRP Cre and POMC Cre mice, we crossed these mice to Lep ob/+ (ob/+, #000632) mice on a C57BL/6 background. Double heterozygous offspring were then crossed to generate AgRP Cre or POMC Cre mice on an Lep ob/bo (ob/ob) background with ob/+ littermates used as controls. For channelrhodopsin-2 expression in AGRP neurons, Agrp Cre mice were crossed with 129S-Gt(ROSA)26Sor tm32(CAG-COP4∗H134R/EYFP)Hze (ROSA26-loxStoplox-ChR2-eYFP, #012569) to generate double mutant animals (AgRP ChR2 ). For channelrhodopsin-2 expression in AgRP neurons on a leptin deficient background, AgRP ChR2 animals were crossed with AgRP Cre ;ob/+ mice. The offspring were then crossed to generate AgRP ChR2 mice on an ob/ob background and ob/+ littermate controls. No statistical methods were used to determine sample sizes. Male and female mice ranging from 8-20 weeks were used. Animals used in intragastric infusion experiments were individually housed, and a cohort of 7 animals was used for experiments with nutrient infusion. A separate group of 4 animals was used for antagonist studies. All other mice were group-housed.

Method Details

Stereotaxic surgery Cre, POMCCre, AgRPCre; ob/ob, AgRPCre; ob/+, POMCCre; ob/ob, and POMCCre; ob/+ mice. During the same surgery a commercially available photometry cannula (Doric Lenses; MFC_400/430-0.48_6.1mm_MF2.5_FLT) or a custom-made photometry cannula (Thorlabs BFH48-400, F112, CF440-10; Chen et al., 2015 Chen Y.

Lin Y.C.

Kuo T.W.

Knight Z.A. Sensory detection of food rapidly modulates arcuate feeding circuits. For photometry experiments, we used recombinant AAV expressing cre-dependent GCaMP6s (AAV1.CAG.Flex.GCaMP6s, Penn Vector Core). AAV was stereotaxically injected unilaterally above the arcuate nucleus of AgRP, POMC, AgRP; ob/ob, AgRP; ob/+, POMC; ob/ob, and POMC; ob/+ mice. During the same surgery a commercially available photometry cannula (Doric Lenses; MFC_400/430-0.48_6.1mm_MF2.5_FLT) or a custom-made photometry cannula (Thorlabs BFH48-400, F112, CF440-10;) was implanted unilaterally in the ARC at the coordinates x = −0.3mm, y = −1.85mm, z = −5.8mm from bregma for AgRP mice and x = −0.3, y = −1.75, z = −5.8 from bregma for POMC mice. For any given set of experiments the same kind of cannula was used. Mice were allowed 2–4 weeks for viral expression and recovery from surgery before photometry recording, mini-osmotic pump implantation or intragastric catheter implantation. For optogenetic experiments, custom-made fiberoptic implants (Thorlabs; 0.39 NA Ø200 mm core FT200UMT and CFLC230-10) were placed unilaterally above the arcuate nucleus of AgRPChR2; ob/ob and AgRPChR2; ob/+ mice at the coordinates x = −0.25 from bregma, y = −1.7 from bregma, z = −5.6 to −5.7 from dorsal skull surface. Mice were allowed 2–3 weeks recovery from surgery before behavior experiments or mini-osmotic pump implantation.

Intragastric catheter implantation Ueno et al., 2012 Ueno A.

Lazaro R.

Wang P.Y.

Higashiyama R.

Machida K.

Tsukamoto H. Mouse intragastric infusion (iG) model. Intragastric catheters were made and implanted as described in detail previously (). Catheters were constructed by attaching 8cm of Silastic tubing (Silastic, Cat 508-003) and 8 cm Tygon tubing (Tygon, AAD04119) to opposite ends of a curved metal connector (Component Supply Company, NE-9019). A 1 cm circle of biologically compatible mesh (gifted by Raul Lazaro) was attached to the silastic tubing 2.3-2.8 cm distal to the edge of the metal connector using adhesive (Xiameter RTV-3110 base and Dow Corning 4 catalyst). A 1 cm by 1.5 cm oval of felt was affixed to the silastic tubing at the distal edge of the the curve in the connector and a 0.5 cm by 1 cm strip of felt was affixed around the metal connector on the proximal edge of its curve. A luer adaptor was placed into the free end of the Tygon tubing (Instech, LS20). Assembled catheters were sterilized using ethylene oxide. AgRPCre mice with functional photometry implants were anesthetized with ketamine/xylazine and the surgical areas shaved and scrubbed with betadine and alcohol. A skin incision of about 1 cm was made between the scapula and the skin dissected from the subcutaneous tissue toward the left flank. A midline abdominal skin incision about 1.5 cm was made extending from the xyphoid process caudally and the skin was dissected from the subcutaneous tissue toward the left flank to complete a subcutaneous tunnel between the two incisions. A hemostat was used to pull the sterilized catheter through the tunnel. The linea alba was incised and the abdominal cavity entered. A small incision was made in the left lateral abdominal wall through which the intragastric catheter was passed into the abdominal cavity. The stomach was externalized and a small puncture made using a jeweler’s forcep. The tip of the cathether was immediately placed into the puncture site and sutured into place using the felt circle with polypropylene suture (CP Medical 8695P). Saline injection into catheter confirmed absence of leakage. The stomach was placed back in the abdominal cavity which was washed with sterile saline. The abdominal muscle was sutured and the skin incision closed in two layers. Next, the catheter was secured at its interscapular site with sutures into the felt oval and surrounding muscle. Finally, the interscapular skin incision was closed. Post-operatively, mice were treated with enrofloxacin, normal saline, and buprenorphine and allowed 7-10 days to recover prior to intragastric infusion and photometry experiments.

Mini-osmotic Pump Implantation Mini-osmotic pumps with a release rate of about 0.5 uL/hr (Alzet, Model 2002) were filled with vehicle or leptin to achieve release of 450 ng leptin per hour. These pumps were implanted subcutaneously into the dorsum of mice. An incision was made and a subcutaneous pocket created by tissue spreading. The pump was placed in this pocket and the skin wound closed with sutures.

Fiber photometry Chen et al., 2015 Chen Y.

Lin Y.C.

Kuo T.W.

Knight Z.A. Sensory detection of food rapidly modulates arcuate feeding circuits. Gunaydin et al., 2014 Gunaydin L.A.

Grosenick L.

Finkelstein J.C.

Kauvar I.V.

Fenno L.E.

Adhikari A.

Lammel S.

Mirzabekov J.J.

Airan R.D.

Zalocusky K.A.

et al. Natural neural projection dynamics underlying social behavior. Two rigs for performing fiber photometry recordings were constructed following basic specifications previously described with minor modifications (). A 473 nm laser diode (Omicron Luxx) was used as the excitation source. This was placed upstream of an optic chopper (Thorlabs MC2000) that was run at 400 Hz. Laser was then split with beam splitter (Thorlabs CM1BS013). Split laser beams each bounced through two kinetic mirrors (Thorlabs BB1-E02, KM100) to allow adjustment of light path. Each laser beam was passed through a GFP excitation filter (Thorlabs MF469-35), reflected by a dichroic mirror (Semrock FF495-Di03-25x36) and coupled through a fiber collimation package (Thorlabs F240FC-A) into a home-made patchcord made with optical fiber (400 μm, 0.48 NA; Thorlabs BFH48-400) or a commercially available patchcord (Doric Lenses, MFP_400/430/1100-0.48_2m_FCM-MF2.5). Patchcords were not changed within experiments. The patchcords were then linked to a fiberoptic implants through ceramic (FIS F18300SSC25) or bronze (Doric Lenses, SLEEVE_BR_2.5) splitting sleeves. Fluorescence outputs were each filtered through a GFP emission filter (Thorlabs MF525-39) and focused by convex lenses (Thorlabs LA1255A) onto photoreceivers (Newport 2151). The signals were output into lock-in amplifiers (Stanford Research System, SR810) with time constant at 30 ms to allow filtering of noise at higher frequency. Those two lock-in amplifiers receive frequency signal of chopper split by BNC splitter. Signals were then digitized with a LabJack U6-Pro and recorded using software provided by LabJack ( https://labjack.com/support/software ) with 250 Hz sampling rate. To reduce photobleaching during recordings that exceeded 3 hr, the laser was modulated as 1 s pulse every 10 s by a TTL signal generator (Graphic State software, Habitest H03-14). A copy of this TTL signal is also sent to LabJack U6-Pro. Each pulse was then extrapolated into a single data point by calculating the median of the center 50%. All experiments were performed in operant chambers (Coulbourn H10-11M-TC) inside a sound-attenuating cubicle (Med Associates ENV-022MD). Experiments were performed during the dark cycle in a dark environment. Mice that didn’t show significant baseline calcium transient, ghrelin response or sensory response to peanutbutter or chow were assumed to be technical failures and were excluded from further experiments or further analysis. To ensure consistent quality of data acquisition, we put the mating sleeve (FIS F18300SSC25) on the implanted cannula after surgery. The sleeve serves as a shield of implanted fiberoptic from scratches and also a gripping point for us to connect the mouse to the photometry rig with enough force to push fiberoptic ends together. Of note, we did not anesthetize the mice before connecting them to the photometry rig. To minimize contamination of the signal by dust in the light path, we cleaned the fiberoptic on the mouse with connector cleaning sticks (MCC25) and precision fiber cleaning fluid (Thorlabs FCS3) or 70% ethanol before each recording. A syringe needle was used to pick out debris which occasionally became stuck in the sleeve. For comparison of data across different days, we let the mouse express virus for at least 6 weeks prior to experiments, which allows GCAMP expression to stabilize. We also refrained from re-aligning light path of photometry rig to ensure consistency.

Intragastric infusions Nutrients at the indicated concentrations or water were infused via ingragastric catheters using a syringe pump (Harvard Apparatus, 70-2001). All infusions were delivered at 50 μL per min with a total infusion volume of 1.2 mL. All photometry experiments involving intragastric infusion were performed in fasted animals unless otherwise specified. Animals were habituated to behavioral chambers for 20 min during photometry recording. During this time, the intragastric catheter was attached to the syringe pump using plastic tubing and adapters (Tygon, AAD04119; Instech, LS20). Total infusion time was 24 min for all experiments. Photometry recording was continued for 15 min after the end of infusion before animals were presented with chow (PicoLab 5058) and then for 20 min following chow presentation. One to three trials of the same experiment for each mouse were combined, averaged, and treated as a single replicate. For peristimulus plots, time zero was defined as the moment that the infusion pump started. All nutrients were diluted into deionized water fresh for each experiment. Vanilla Ensure powder was dissolved at a concentration of 0.42 g/mL of solution; 20% intralipid (Sigma-Aldrich) was used both undiluted and diluted to 6.4%; premium collagen peptides (Sports Research) was dissolved at concentrations of 0.45 g/mL and 0.15 g/mL; glucose was dissolved at 0.45 g/mL, 0.24 g/mL, 0.12 g/mL, and 0.06 g/mL; sucrose, fructose, and galactose were dissolved at 0.24 g/mL; and sucralose was dissolved at 8 mg/mL.

Drug and hormone injections Hormones and small molecules were injected at the concentrations and routes indicated below during photometry recording. All compounds were injected at a volume of 10 uL/g body weight. Animals were habituated to the recording chambers for 20 min prior to injection. Following hormone injection, photometry recording continued for 35 min or longer as indicated. For the combination injection of leptin and CCK, leptin was injected 2 hr prior to the start of recording, and then animals were habituated and injected with CCK as described for other experiments. For other hormone combinations, both hormones were injected simultaneously after habituation to the recording chamber. One to three trials of the same experiment for each mouse were combined, averaged, and treated as a single replicate. For peristimulus plots, time zero was defined as the moment that the investigator opened the behavioral chamber. Essner et al., 2017 Essner R.A.

Smith A.G.

Jamnik A.A.

Ryba A.R.

Trutner Z.D.

Carter M.E. AgRP neurons can increase food intake during conditions of appetite suppression and inhibit anorexigenic parabrachial neurons. To evaluate the effect of LPS on feeding and on sensory inhibition of AgRP neurons, we presented LPS- or PBS-treated animals with chow 4 hr after injection (). Fasted mice were injected with LPS or PBS 3.5 hr prior to placement in the recording chambers. After 30 min of recording animals were presented with chow (PicoLab 5058). Photometry recording continued for 20 min after chow presentation after which the quantity of chow consumed was measured. One trial each of LPS and PBS was performed per mouse in an LPS-naive cohort. We used the following doses based on previously published reports, unless otherwise specified. Glucose 4.5 g/kg IP (Sigma), CCK octapeptide 10 ug/kg IP (Bachem), serotonin hydrochloride 2 mg/kg IP (Sigma-Aldrich), PYY 0.1 mg/kg IP (R&D Systems), leptin 2 mg/kg IP (R&D Systems), liraglutide 0.4 mg/kg IP (Novo Nordisk; generous gift from Dr. Randy Seeley), amylin 10 ug/kg IP (Tocris), glucagon 2 mg/kg SQ (Bachem), lithium chloride 84 mg/kg IP (Acros), LPS 100 ug/kg (Sigma), and ghrelin 2 mg/kg IP (R&D Systems). All of these compounds were dissolved in saline except for glucose which was dissolved in water.

Antagonist studies Shoblock et al., 2010 Shoblock J.R.

Welty N.

Nepomuceno D.

Lord B.

Aluisio L.

Fraser I.

Motley S.T.

Sutton S.W.

Morton K.

Galici R.

et al. In vitro and in vivo characterization of JNJ-31020028 (N-(4-4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a selective brain penetrant small molecule antagonist of the neuropeptide Y Y(2) receptor. To observe the effects of devazepide and ondansetron on baseline AgRP neuron activity, these antagonists or vehicle controls were injected via intragastric catheters into fasted mice after 20 min of habituation to the behavioral chamber during photometry recording. 3-5 min after drug injection, animals received intragastric infusion of water, which has no effect on AgRP neuron activity. AgRP neuron activity in antagonist-injected mice was compared to vehicle-injected controls (devazepide) or to animals who received only water infusion (ondansetron) at a time point 25 min after antagonist injection. To observe the effects of JNJ-31020028 on AgRP neuron activity, this drug was injected subcutaneously into fasted mice after 20 min of habituation to the behavioral chamber during photometry recording. This route was chosen because this drug is not orally bioavailable (). Signal from these animals was compared to signal from vehicle-injected mice 25 min after injection. For peristimulus plots, time zero was defined as the moment that the investigator opened the behavioral chamber. Shoblock et al., 2010 Shoblock J.R.

Welty N.

Nepomuceno D.

Lord B.

Aluisio L.

Fraser I.

Motley S.T.

Sutton S.W.

Morton K.

Galici R.

et al. In vitro and in vivo characterization of JNJ-31020028 (N-(4-4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a selective brain penetrant small molecule antagonist of the neuropeptide Y Y(2) receptor. To observe the effects of antagonists on the response of AgRP neuron activity to nutrient infusion, animals were habituated to the recording chambers for 20 min prior to antagonist delivery. For devazepide and ondansetron, intragastric infusion of lipid, glucose, or water was started 3-5 min after antagonist delivery. For JNJ-31020028, intragastric infusion of lipid, glucose, or water infusion was started 25-30 min after antagonist delivery, since this is the time at which drug reaches maximum serum concentration (). Infusion time was 24 min for all experiments. Photometry recording was continued for 15 min after infusion. For peristimulus plots time zero was defined as the moment that the infusion pump was started. Doses were chosen based on previously published reports: devazepide 1 mg/kg IG (R&D Systems) was diluted in 5% DMSO, 5% Tween 80 in PBS, ondansetron 1 mg/kg IG (Sigma) was diluted in normal saline, and JNJ-31020028 10 mg/kg SC (MedKoo Biosciences) was diluted in 5% DMSO, 5% Tween 80 in PBS. All compounds were injected at a volume of 10 uL/g body weight.

Food and Object Presentation To eliminate any effects of novelty, mice were exposed prior to testing to peanut butter, chocolate, and “cages” as described in the main text. Mice were either fasted overnight (16 hr) or fed ad libitum, acclimated to the behavioral chamber, and then presented with chow, peanut butter, caged chocolate, or available chocolate as indicated in the main text. For peristimulus plots time zero was defined as the moment that the investigator opened the behavioral chamber.