Animals

Male Sprague Dawley rats (Envigo, Indianapolis, IN, USA) weighing 300–400 g were individually housed in wire-hanging cages in a climate controlled (22–24 °C) environment with a 12:12 h light/dark cycle. Except where noted, rats were given ad libitum access to water and standard rodent chow (LabDiet 5001, LabDiet, St. Louis, MO). Experiments were performed in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, and all procedures were approved by the Institutional Animal Care and Use Committee of the University of Southern California.

Stereotaxic cannula implantation

Rats were first anesthetized with an intraperitoneal injection of ketamine (90 mg/kg)/xylazine (2.8 mg/kg)/acepromazine (0.72 mg/kg), prepped for surgery, and placed in a stereotaxic apparatus. For ICV cannulae, unilateral guide canulae (26-gauge, Plastics One) were surgically implanted targeting the lateral ventricle using the following coordinates57: −0.9 mm anteioror/posterior (AP), +1.8 mm medial/lateral (ML), −2.6 mm dorsal/ventral (DV) (0 reference point for AP and ML at bregma, 0 reference point for DV at skull surface at target site). Placement for the lateral ventricle cannula was verified by elevation of cytoglucopenia resulting from an injection of 210 μg (2 μl) of 5-thio-d-glucose (5tg)58 using an injector that extended 2 mm beyond the end of the guide cannula. A postinjection elevation of at least 100% of baseline glycemia was required for subject inclusion. Animals that did not pass the 5tg test were retested with an injector that extended 2.5 mm beyond the end of the guide cannula and, upon passing 5tg, were subsequently injected using a 2.5-mm injector instead of a 2-mm injector for the remainder of the study.

For targeting the vHP, bilateral cannulae (26-gauge, Plastics One, Roanoke, VA) were implanted at the following stereotaxic coordinates57: −4.9 mm AP, +/−4.8 mm ML, −6.1 mm DV. Injectors for drug administration projected 2 mm beyond the guide cannulae. Placements for vHP cannulae were verified postmortem by injection of blue dye (100 nl, 2% Chicago sky blue ink) through the guide cannulae. Data from animals with dye confined to the vHP were included in the analyses.

Immunohistochemistry

Rats were anesthetized and sedated with a ketamine (90 mg/kg)/xylazine (2.8 mg/kg)/acepromazine (0.72 mg/kg) cocktail, then transcardially perfused with 0.9% sterile saline (pH 7.4) followed by 4% paraformaldehyde (PFA) in 0.1 M borate buffer (pH 9.5; PFA). Brains were dissected out and post-fixed in PFA with 15% sucrose for 24 h, then flash frozen in isopentane cooled in dry ice. Brains were sectioned to 30-µm thickness on a freezing microtome. Sections were collected in 5 series and stored in antifreeze solution at −20 °C until further processing.

General fluorescence IHC labeling procedures were performed29. The following antibodies and dilutions were used: rabbit anti-MCH (1:1000; Phoenix Pharmaceuticals, Burlingame, CA, USA), rabbit anti-RFP (1:2000, Rockland Inc., Limerick, PA, USA), and Guinea Pig anti FG (1:5000; Protos Biotech Corp., New York, NY, USA). Antibodies were prepared in 0.02 M potassium phosphate-buffered saline (KPBS) solution containing 0.2% bovine serum albumin and 0.3% Triton X-100 at 4 °C overnight. After thorough washing with 0.02 M KPBS, sections were incubated in secondary antibody solution. All secondary antibodies were obtained from Jackson Immunoresearch and used at 1:500 dilution at 4 °C, with overnight incubations (Jackson Immunoresearch; West Grove, PA, USA). Sections were mounted and coverslipped using 50% glycerol in 0.02 M KPBS and the edges were sealed with clear nail polish.

IHC detection of MCH was performed according to the following sequence (overnight incubations on a motorized rotating platform at 4 °C): Sections were removed from anti-freeze and washed in [1] 0.02 M KPBS (6 changes in 2 h), [2] KPBS with 0.3% hydrogen peroxide (15 min), [3] KPBS (3 changes), [4] KPBS with 0.3% Triton X-100 (45 min), [5] KPBS (3 changes), [6] KPBS with 2% donkey serum (10 min), [7] KPBS with 1% donkey serum, 0.1% Triton X-100, and rabbit anti-MCH antibodies [1:2000; rabbit anti-MCH]17. Primary antibody incubation length was ~60 h. [8] KPBS (8 changes in 2 h), [9] KPBS with 0.1% Triton X-100 and biotinylated secondary antibodies (1:1000; biotinylated donkey anti-rabbit, Jackson Immunoresearch; overnight). [10] KPBS (6 changes), [11] KPBS with tertiary reagent (1:1000 of reagent A and B from ABC Elite kit, Vector Labs; 4 h), [12] KPBS (3 changes), [13] KPBS with 0.05% 3,3′-diaminobenzidine (Sigma) and 0.005% H 2 O 2 (15 min), [14] KPBS (4 changes). Sections were then mounted, air-dried, dehydrated with ascending concentrations of alcohol solutions, cleared in xylene, and coverslipped with DePeX mounting medium.

Photomicrographs were acquired using either a Nikon 80i (Nikon DS-QI1,1280X1024 resolution, 1.45 megapixel) under epifluorescence or darkfield illumination or as optical slices using a Zeiss LSM 700 UGRB Confocal System (controlled by Zeiss Zen software).

Intracranial virus injections

For stereotaxic injections of viruses and tracers, rats were first anesthetized and sedated with a ketamine (90 mg/kg)/xylazine (2.8 mg/kg)/acepromazine (0.72 mg/kg) cocktail. Animals were shaved, surgical site was prepped with iodine and ethanol swabs, and animals were placed in a stereotaxic apparatus for stereotaxic injections. Viruses were delivered using a microinfusion pump (Harvard Apparatus, Cambridge, MA, USA) connected to a 33-gauge microsyringe injector attached to a PE20 catheter and Hamilton syringe. Flow rate was calibrated and set to 5 µl/min; injection volume was 200 nl/site. Injectors were left in place for 2 min postinjection. Following injections, animals were either sutured or surgically implanted with a cannula where described. All experimental procedures occurred 21 days post virus injection to allow for transduction and expression. Successful virally mediated transduction was confirmed postmortem in all animals via IHC staining using immunofluorescence-based antibody amplification to enhance the fluorescence followed by manual quantification under epifluorescence illumination using a Nikon 80i (Nikon DS-QI1,1280X1024 resolution, 1.45 megapixel).

Designer receptors exclusively activated by designer drugs

Bilateral stereotaxic injections of AAV2-rMCHp-hM3D(Gq)-mCherry or AAV2-DIO-rMCHp-hM3D(Gq)-mCherry were made at the following coordinates57: injection (1) −2.6 mm AP, ±1.8 mm ML, −8.0 DV; (2) −2.6 mm AP, ±1.0 mm ML, −8.0 DV; (3) −2.9 mm AP, ±1.1 mm ML, −8.8 DV; (4) −2.9 mm AP, ±1.6 mm ML, −8.8 DV (from the skull surface at bregma).

For the dual-virus approach for selective expression of DREADDs in MCH neurons that project to the vHP, bilateral injections of the canine adenovirus 2 Cre (CAV2 CRE; 200 nl) were delivered using the following coordinates57: −4.9 mm AP, 4.8 mm ML, −7.8 mm DV (from the skull surface at the injection site) prior to injections with the MCH AAV2-DIO-rMCHp-hM3D(Gq)-mCherry.

Characterization of DREADD expression

DREADD expression was quantified in 1 out of 5 series of brain tissue sections from the perfused brains cut at 30 µm on a freezing microtome based on counts for the fluorescence reporter mCherry. Immunofluorescence staining for red fluorescent protein (RFP) was conducted as described above to amplify the mCherry signal. Counts were performed in sections from Swanson Brain Atlas level 27–3239, which encompasses all MCH-containing neurons. Cell counts were performed in all DREADD virus-injected animals. For MCH DREADD experiments, animals were excluded from all experimental analyses if fewer than 2/3 of the total number of MCH neurons were transduced with RFP (based on IHC staining for MCH). For the dual-virus cre-dependent MCH DREADD experiments, all animals were included in the experimental analyses. Counts were performed by 2 researchers using epifluorescence illumination using a Nikon 80i (Nikon DS-QI1,1280X024 resolution, 1.45 megapixel) and the average of the 2 counts was taken. Researchers who performed the counting were kept consistent between cohorts and blind to experimental assignments.

AAV-mediated RNA interference for MCHR1

A custom shRNA targeting MCHR1 mRNA was cloned and packaged into an AAV1 under the control of a U6 promoter and co-expressing GFP (AAV1-GFP-U6-r-MCHR1-shRNA; Vector Biolabs, Malvern, PA, USA). For screening, 4–5 shRNA candidates were transfected into HEK293 cells to compare the knockdown efficiency for each shRNA. A reporter assay was used to assess the knockdown efficiency of each shRNA candidates, and the best shRNA was used to make the AAV virus. The shRNA was validated in vitro for ~95% knockdown of the mRNA for PMCH. This virus is now commercially available from Vector Biolabs (Malvern, PA, USA) upon request. The sequence is as follows:

5′-CACC GGAGTGTCTCCTACATCAACAC TCGAG TGTTGATGTAGGAGACACTCC-TTTTT-3′

The targeting sequence is GGAGTGTCTCCTACATCAACA and the hairpin loop sequence is CTCGAG. A separate AAV1 containing shRNA targeting a scrambled nonsensical sequence along with GFP was used as a control. AAVs or artificial cerebrospinal fluid (aCSF) were delivered bilaterally to the vHP (AP: −4.9, ML: +/−4.8, DV: −7.8, with DV zero at the skull at the injection site) at an injection volume of 200 nl via pressure injection using the stereotaxic procedures described above. The titer for the MCHR1 shRNA was 3.3 × 1013 GC/ml and for the scrambled shRNA 1.7 × 1013 GC/ml.

Neural pathway tracing

For retrograde pathway tracing, rats were anesthetized and sedated with a ketamine (90 mg/kg)/xylazine (2.8 mg/kg)/acepromazine (0.72 mg/kg) cocktail and placed in a stereotaxic apparatus. Rats received a unilateral iontophoretic injection of FG (Fluorochrome LLC; 2% in 0.9% NaCl) targeting either vHP (n = 4): −4.7 mm AP, 4.6 mm ML, −6.4 mm DV (from the dura at the injection site) or the ACB (n = 4): 1.2 mm AP, 1.0 mm ML, −6.75 mm DV (from the dura at the injection site) (coordinates from ref. 57). Iontophoresis was performed using a precision current source (Digital Midgard Precision Current Source, Stoelting, Wood Dale, IL, USA) as described previously14. Following a 12-day survival period, animals were fixation-perfused and tissue was harvested and processed for immunofluorescence.

Fluorescence in situ hybridization

Tissue sections were obtained as in Immunofluorescence and mounted on subbed glass slides (Fisher brand Superfrost Plus, Fisher Scientific, Hampton, NH, USA) and desiccated overnight (~16 h) in a vacuum desiccant chamber. Following 1 h and 45 min postfix in 4% PFA, sections were washed 5 × 5 min in KPBS and incubated for 30 min at 37 °C in a solution of 100 mM Tris (pH 8), 50 mM EDTA (pH 8), and 0.1% Proteinase K (10 mg/ml, Sigma P2308), then rinsed for 3 min in the same Tris and EDTA solution without Proteinase K. Sections were washed 3 min in a solution of 100 mM triethanolamine (pH 8) in water and then incubated for 10 min at room temperature with 0.25% acetic anhydride in 100 mM triethanolamine, then washed 2 × 2 min in 10% 20× saline-sodium citrate buffer. Prior to hybridization, sections were dehydrated in increasing concentrations of ethanol (50%, 70%, 95%, 100%, 100%). Sections were incubated with probes for 3 h (MCHR1, ACD Cat #413191; vGLUT1, ACD Cat #317001; GAD2, ACD Cat #435801). Reagents from the RNAscope® Fluorescent Multiplex Detection Reagent Kit v2 (Advanced Cell Diagnostics, Newark, CA, USA; Cat #: 323100) were used to amplify the probe as per the kit’s instructions. Slides were coverslipped using ProLong® Gold Antifade Reagent (Cell Signaling, Danvers, MA, USA; Cat #: 9071s). Photomicrographs were acquired using a Nikon 80i (Nikon DS- QI1,1280X1024 resolution, 1.45 megapixel) under epifluorescent illumination using the Nikon Elements BR software.

Drug preparation and intracranial pharmacological injection

For ICV and vHP injections of MCH, MCH (Bachem Americas, Torrance, CA, USC; Cat #: H-2218.1000) was dissolved in aCSF and diluted to 0.5 µg/µl (for vHP injections) or 5 µg/µl (for ICV injection), except where noted. For chemogenetic activation of MCH neurons, CNO (National Institute of Mental Health; 18 mmoles in 2 μl) or 33% dimethyl sulfoxide (DMSO) in aCSF (daCSF; vehicle control in 2 μl) was administered ICV. Animals were handled and habituated to injections prior to testing. All injections were delivered through a 33-gauge micro-syringe injector attached to a PE20 catheter and Hamilton syringe. For vHP injections, a microinfusion pump (Harvard Apparatus) was used. The flow rate was set to 5 μl/min and 100 nl injection volume. Injectors were left in place for 30 s to allow for complete infusion of the drug. For ICV injections, 1 µl (MCH and aCSF) or 2 µl (CNO and daCSF) was delivered by manually plunging the Hamilton syringe.

Differential reinforcement of low rates of responding

The protocol that we used for the differential reinforcement of low rates of responding task (DRL) was modified from refs. 59,60,61. Rats were first habituated in their home cage to 45 mg pellets containing 35% kcal from fat with sucrose (F05989, Bio-Serv, Frenchtown, NJ, USA). On each day of DRL training, chow was removed from the home cage 1 h prior to training, which began at the onset of the dark cycle, and chow was returned to the animals following DRL training. For DRL, animals were placed in an operant chamber (Med Associates, Fairfax, VT, USA) containing an active (reinforced with 1 palatable 45 mg pellet, with stimulus light activated during a reinforced lever press) and inactive (non-reinforced) lever. The task is 45 min long (one session per treatment), during which time the levers are extended, and the animals may respond as many times as they choose for the entire 45 min. For the first 5 days of training, animals were on a DRL0 schedule, where each active lever press is reinforced with a 0-s time delay. Animals were then switched to a DRL5 schedule for 5 days, where rats must withhold pressing for a 5-s interval after pressing for each subsequent lever press to be reinforced, followed by 5 days of DRL10 (10 s withholding period) and 10 days of DRL 20 (20 s withholding period). A 2-day rest period occurred in between each 5 days of training. Efficiency in the DRL task was calculated as the number of pellets earned/the number of active lever presses.

On test days, food was removed 2 h prior to the lights going off and behavioral testing began at lights offset. For investigating the effects of MCH injection on DRL, animals were randomized to receive either aCSF or MCH using a counterbalanced within-subjects design, with 72 h between treatments 1 and 2. Injections were given 45 min prior to behavioral testing. For the effects of chemogenetic activation of MCH neurons on performance in DRL, animals were randomized to receive either daCSF or CNO using a counterbalanced within-subjects design with 72 h between treatments 1 and 2. CNO or daCSF were given 1.5 h prior to behavioral testing.

Food intake studies

A separate cohort of rats was used for each feeding experiment with food intake analyses occurring in the animal’s home cage. For both studies using laboratory chow (LabDiet 5001,13% fat, 29% protein, 58% carbohydrate by kcal, LabDiet, St. Louis, MO, USA) and high-fat diet (Research Diets D12451, 45% fat, 20% protein, 35% carbohydrate by kcal, Research Diets Inc., New Brunswick, NJ, USA), home cage food was removed 2 h prior to the lights going off. Animals were randomized to receive either aCSF, 0.5 µg, or 1 µg MCH (for vHP studies) or aCSF, 5 µg, or 10 µg MCH (for ICV studies) in a counterbalanced within-subjects design. Injections were given 45 min prior to the lights going off and pre-weighed amounts of the test diet were deposited in the home cage immediately after the lights went out. For chemogenetic activation of MCH neurons, rats were randomized to receive either CNO (National Institute of Mental Health; 18 mmol in 2 μl) or 33% DMSO in aCSF (daCSF; vehicle control in 2 μl) ICV. Injections were given 1.5 h prior to the lights going off and pre-weighed amounts of the test diet were deposited in the home cage immediately after the lights went out. Spill papers were placed underneath the cages to collect food crumbs. Food spillage was weighed and added to the difference between the initial hopper weight and the hopper weight at each measurement time point. A total of 72 h was allotted between treatments.

Delay discounting task

The protocol for the delay discounting task was modified from refs. 33,62. Rats were first habituated to palatable 45 mg sucrose pellets (F0023, Bio-Serv, Frenchtown, NJ, USA) in their home cage, then trained using a fixed ratio-1 (FR1) schedule to lever press for palatable 45 mg sucrose pellets in an operant chamber (Med Associates). On each day of the initial training, only the right or the left lever were retracted (in a counterbalanced fashion) and training continued until the animal reached the criterion of 50 lever presses in 45 min on both levers. Animals were then trained in a series of 4 blocks of 5 choice trials, where one lever consistently delivered 1 pellet per lever press, and the other lever delivered 4 pellets per press in a time delay of 0, 15, 30, and 45 s corresponding to block 1, 2, 3, and 4. Each block began with a forced trial on each lever, in random order. A stimulus light above each lever was lit during the forced trial and during the choice trials when the levers were extended. For each choice trial, levers remained extended for 10 s or until a lever response was made, after which the levers were retracted and the stimulus light remained illuminated over the pressed lever until pellets were dispensed. Preference for the smaller immediate reward over the larger, delayed reward is indicative of delay discounting.

On test day, food was removed 2 h prior to the lights going off and behavioral testing began at lights offset. Animals were randomized to receive either aCSF or MCH using a counterbalanced within-subjects design, with a washout period of 72 h between treatments 1 and 2. Injections were given 45 min prior to behavioral testing, and food was returned after the testing was complete.

Open field test

The apparatus used for the open field test is an opaque gray plastic bin (60 cm × 56 cm), which was positioned on a flat table in an isolated room with a camera directly above the center of the apparatus. Desk lamps were positioned to deliver indirect light on all corners of the maze such that the lighting in the box measured 30 lux in all corners and in the center. At the start of the 10-min test, each animal is placed in the open field apparatus in the same corner facing the center of the maze. All sessions were video recorded and ANY-maze video tracking software (Stoelting, Wood Dale, IL) was used for activity tracking. Total distance traveled was measured by tracking movement from the center of the rat’s body.

On test days, food was removed 2 h prior to the lights going off and behavioral testing began when the lights turned off. For investigating the effects of MCH injection on open field activity levels, animals were randomized to receive either aCSF or MCH using a counterbalanced within-subjects design, with a washout period of 72 h between treatments 1 and 2. Injections were given 45 min prior to behavioral testing.

Fixed interval (FI) and peak interval task

Following recovery from surgery, rats (n = 6) were habituated for 15 min in their home cage to 20% sucrose solution. Rats were then acclimated to the conditioning boxes for two sessions where they received a total of 32, 0.1 ml sucrose deliveries to a recessed food cup, presented on a variable time (VT) 240 s schedule. Subsequently they received 10 sessions of FI training. In these sessions, during each trial (presented on a VT 60 s schedule) a lever was extended and the house light was illuminated. Each response to the lever was reinforced on a FI 20 s schedule resulting in sucrose delivery and occurred contemporaneously with both the retraction of the lever and the termination of the house light. Each session was completed after either 120 min had elapsed or 50 reinforcers had been delivered. During each of the remaining 20 sessions, rats received random presentation of 25 FI 20 s trials and 25 unreinforced probe trials. In each probe trial, the lever and house light were presented for 60 s followed by an added random period of time with a mean of 20 s driven by a Gaussian distribution. At the termination of the trial, the lever and the house light were retracted and extinguished, respectively. For the final four sessions, rats received 2 μl ICV infusions of either 18 mmol CNO or 0.2 M PBS, 15 min prior to the start of the session. Data from these test days were combined, resulting in 50 peak interval probe trials under chemogenetic stimulation via CNO and 50 trials under control PBS conditions.

PR task

The PR protocol was modified from ref. 63. Training occurred in operant conditioning boxes (Med Associates; Fairfax, VT, USA) in 1 h sessions over 6 days. An FR1 with autoshaping procedure was used during the initial 2 days, where each lever press was reinforced with a 45-mg pellet (35% kcal from fat enriched with sucrose, F05989, Bio-Serv, Frenchtown, NJ). For the autoshaping component, a pellet was dispensed every 600 s that elapsed without operant-based reinforcement. The animals then received 2 days of FR1 schedule with no autoshaping, followed by 2 days of FR3 training, where 3 presses were required for each pellet earned. For all procedures, there was an active (reinforced) and an inactive (non-reinforced) lever. On test day, the response requirement of the PR schedule increased progressively as previously described63,64,65 using the following formula: F(i) = 5e^0.2i–5, where F(i) is the number of lever presses required for next pellet at i = pellet number. The breakpoint for each animal was defined as the final completed lever press requirement that preceded a 20-min period without earning a reinforcer.

On test day, food was removed 2 h prior to the lights going off and behavioral testing began at lights offset. Animals were randomized to receive either aCSF or MCH using a counterbalanced within-subjects design, with a washout period of 72 h between treatments 1 and 2. Injections were given 45 min prior to behavioral testing, and food was returned after the testing was complete.

Quantitative PCR (qPCR) quantification of in vivo MCHR1 knockdown

Brains from sham, scramble, or MCHR1-injected animals were rapidly removed and flash frozen in isopentane and stored at −80 °C. Serial coronal sections (50 μm) of the midbrain/forebrain were mounted on a slide and viewed immediately under a fluorescent microscope (Nikon 80i) until EGFP-expressing neurons were visualized. Total RNA was extracted from 1 mm micropunches of the vHP CA1 region using the Purelink RNA Mini kit (ThermoFisher Scientific, Canoga Park, CA, USA). cDNA was synthesized from 125 ng total RNA using the iScript cDNA Synthesis Kit (BioRad Inc., Hercules, CA, USA) and qPCR was carried out in duplicate using TaqMan Fast Advanced Master Mix (ThermoFisher Scientific). Samples were run using StepOne Plus Real Time System with the primer/probe sets for MCHR1 (Rn00755896_m1) and GAPDH (internal control; Rn0177563_g1) (ThermoFisher Scientific). Relative mRNA expression was calculating using the comparative Ct method.

Functional brain mapping

Autoradiographic 2-DG uptake metabolic mapping was used to access functional brain activation following bilateral injection of MCH into the vHP. Animals were implanted bilaterally with guide cannula for drug injection targeting the ventral CA1 as described above and allowed to recover for at least 3 weeks before the mapping experiment. Animals were individually housed and randomly assigned into one of the two groups: MCH (n = 9, body weight 411 ± 20 g on the day of mapping) and vehicle (n = 8, body weight 414 ± 20 g). For 3 days prior to the mapping experiment, animals were acclimated each day to handling (5 min), the experiment room (15 min), and a cylindrical, plexiglass experimental arena (30 min, diameter = 30 cm, height = 30 cm) under low-level ambient lighting.

Relative rCGU was measured according to the method of Sokoloff66 with modification67. On the day of mapping, food was removed 120 min before the injection of 2-DG to prevent interference with glucose uptake by food consumption. Seventy minutes after food removal, the animals were injected bilaterally with MCH solution (5 μg/μl in aCSF) or vehicle (aCSF) through an injection cannulae projected 2 mm beyond the guide cannulae, at a 5 μl/min rate and 100 nl injection volume per hemisphere. The animal was left in its home cage for 50 min before receiving an IP injection of 2-DG (Moravek Inc., Brea, CA, USA; cat # MC355, 0.1 µCi/g body weight in 0.53 ml saline). The animal was immediately placed inside the arena and left there for 45 min for 2-DG uptake mapping in a no task, resting state. Thereafter, the animal was deeply anesthetized with 4% isoflurane and euthanized by an intracardiac injection of 3 M potassium chloride solution (1 ml).

Brains were extracted, flash frozen in methylbutane on dry ice, and serially sectioned (57 coronal 20-μm slices, 300-μm interslice distance beginning at ~4.5 mm anterior to the bregma) in a cryostat (HM550, Microm International GmbH, Walldorf, Germany). Slices were heat-dried on glass slides and exposed to Kodak Biomax MR films (Eastman Kodak, Rochester, NY, USA) for 3 days at room temperature. Autoradiographic images of brain slices were digitized on an 8-bit gray scale using a voltage-stabilized light box (Northern Lights Illuminator, Interfocus Imaging Ltd., Cambridge, UK) and a Retiga 4000R charge-coupled device monochrome camera (Qimaging, Surrey, Canada). For each animal, a three-dimensional brain was reconstructed from 57 digitized autoradiograms (voxel size: 40 × 300 × 40 μm3) in ImageJ. Adjacent sections were aligned manually in Photoshop (version 9.0, Adobe Systems Inc., San Jose, CA, USA) and using TurboReg, an automated pixel-based registration algorithm implemented in ImageJ (version 1.35, http://rsbweb.nih.gov/ij/).

We and others have adapted the statistical parametric mapping (SPM) package (Wellcome Centre for Neuroimaging, University College London, London, UK) for the analysis of rodent autoradiographic cerebral blood flow68 and CGU data69. For preprocessing, one artifact free brain was selected as reference. All brains were spatially normalized to the reference brain in SPM (version 5). Spatial normalization consisted of applying a 12-parameter affine transformation followed by a nonlinear spatial normalization using three-dimensional discrete cosine transforms. All normalized brains were then averaged to create a final rat brain template. Each original brain was then spatially normalized to the template. Final normalized brains were smoothed with a Gaussian kernel (full-width at half-maximum = 240 × 300 × 240 μm3) to improve the signal-to-noise ratio. Proportional scaling was used to normalize whole-brain average CGU across animals.

Unbiased, voxel-by-voxel Student’s t tests between the MCH and vehicle groups were performed across the whole brain to access changes in rCGU following MCH injection into the ventral CA1. Threshold for significance was set at P < 0.05 at the voxel level and an extent threshold of 200 contiguous voxels. This combination reflected a balanced approach to control both Type I and Type II errors. The minimum cluster criterion was applied to avoid basing our results on significance at a single or small number of suprathreshold voxels. Brain regions were identified according to a rat brain atlas39.

Seed ROI correlation analysis was used to assess functional connectivity of the ventral CA140. Structural ROI of the ventral CA1 was hand drawn in MRIcro (version 1.40, http://cnl.web.arizona.edu/mricro.htm) over the template brain in the left hemisphere according to the rat brain atlas. The structural ROI was then intersected with clusters showing MCH-induced significant increases in rCGU to create a functional seed ROI. Mean optical density of the seed ROI was extracted for each animal using the MarsBaR toolbox for SPM (version 0.42, http://marsbar.sourceforge.net/). Correlation analysis was performed in SPM for each group using the seed values as a covariate. Threshold for significance was set at P < 0.05 at the voxel level and an extent threshold of 200 contiguous voxels. Regions showing significant correlations in rCGU with the seed ROI are considered functionally connected with the seed. Statistical significance of between-group differences in correlation coefficients (between the seed and another ROI) was evaluated using Fisher’s Z-transform test (P < 0.05).

Statistical analyses

Analysis of variance (ANOVA) were performed using the GraphPad Prism 7.0 Software (GraphPad Software Inc., San Diego, CA, USA) and t tests were performed using either GraphPad Prism 7.0 software or Microsoft Excel for Mac (v. 15.26; Microsoft Inc., Redmond, WA, USA). DRL, PR, and shRNA quantification data were analyzed using a Student’s two-tailed paired t test. All comparisons of the effects of either CNO or MCH on chow or high-fat diet intake were analyzed using a repeated-measures ANOVA with Tukey’s post hoc test for multiple comparisons. Delay discounting data were analyzed using a two-way repeated-measures ANOVA with Sidak test for multiple comparisons. For all statistical tests, the α level for significance was 0.05.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.