Further information and requests for resources and reagents should be directed to and will be fulfilled by Michael J. Krashes ( michael.krashes@nih.gov ).

Glp1r-ires-Cre (), Mc4r-T2a-Cre (), Oxt-ires-Cre (), Crh-ires-Cre (), B6.Cg-Gt(ROSA)26Sor/J (Ai6-ZsGreen) (), B6.Cg-Gt(ROSA)26Sor/J (Ai3-EYFP) () mice were generated and maintained as previously described. All mice are on a C57B6 background. To generate reporter mice Glp1r-ires-Cre, Mc4r-T2a-Cre, Oxt-ires-Cre or Crh-ires-Cre animals were crossed with Ai6-ZsGreen or Ai3-EYFP animals. All animal care and experimental procedures were approved by the National Institute of Health 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, 3.3 kcal gmetabolizable energy, 12.5% kcal from fat; Harlan Teklad) and water provided ad libitum. All diets were provided as pellets. For all behavioral studies male and female mice between 6–20 weeks were used. For electrophysiological studies male and female mice between 4–10 weeks were used.

Method Details

Brain Tissue Preparation Mice were terminally anesthetized with chloral hydrate (Sigma Aldrich) and transcardially perfused with phosphate-buffered saline (PBS) followed by 10% neutral buffered formalin (Fisher Scientific). Brains were extracted, cryoprotected in 20% sucrose, and sectioned coronally on a freezing sliding microtome (Leica Biosystems) at 30, 40 or 50 μm and collected in four equal series.

Immunohistochemistry Brain sections were washed in 0.1 M phosphate-buffered saline with Tween-20, pH 7.4 (PBST), blocked in 3% normal goat serum/0.25% Triton X-100 in PBS for 1 hour at room temperature and then incubated overnight at room temperature in blocking solution containing primary antiserum rabbit anti-c-Fos, Millipore-Sigma, 1:1000; rat anti-tdTomato, Kerafast, 1:1000; chicken anti-GFP, Life Technologies, 1:1,000; rabbit anti-oxytocin, Peninsula Laboratories, 1:1000. The next morning sections were extensively washed in PBS and then incubated in Alexa fluorophore secondary antibody (Molecular Probes, 1:1000) for 2 h at room temperature. After several washes in PBS, sections were mounted onto gelatin-coated slides and fluorescent images were captured with an Olympus VS120 slide scanner microscope and/or Zeiss Observer Z1 confocal microscope. DAB Staining Floating sections were incubated with 1% H 2 O 2 in PBS to remove endogenous peroxidase activity. The sections were then washed with PBS and the pre-incubated in a blocking buffer consisting of PBS with 0.3% triton and 3% normal donkey serum. Sections were then incubated with rabbit monoclonal anti-c-fos primary antibody at 1:2000 dilution (Cell Signaling, Cat. 2250,) overnight at room temperature. Sections were washed extensively in PBS and then incubated with biotinylated donkey anti-rabbit antibody (1:4000; Jackson Laboratories, 711-065-152) for 2 hours. c-fos was visualized by a chromogenic reaction using the DAB kit from Thermofisher (Cat. 34065). Slides were then dried and mounted in DPX Mounting medium (Electron Microscopy Science) and images were captured with a Keyence BZ-X710 microscope.

RNAScope In Situ Hybridization (ISH) Mice were deeply anesthetized with isoflurane and brains were harvested and flash frozen in 2-methylbutane. Brains were then cut as 16 μm sections onto glass slides and then stored at -80°C until processed for in situ hybridization using RNAscope technology. The probes were purchased from Advanced Cell Diagnostics, and the assays were performed according to the manufacturer’s protocol. The sections were fixed in cold 10% formalin for 1 hour, followed by dehydration in 50% and 75% ethanol for 5 minutes each, and 100% ethanol twice. Sections were dried at 40C for 30 min before proceeding with hybridization. For fluorescent staining, the RNAscope Fluorescent Multiplex Reagent Kit (320850) was used. The sections were pretreated in protease IV for 30 minutes, washed 2 times in PBS, and incubated with the desired probes for 2 hours at 40C in the EZ Hybridization oven (ACD, 310010). The slides were washed 2 times in 1x wash buffer (ACD, 310091) for 2 minutes each. Sections were incubated with Amp1 for 30 min, Amp2 for 15 min, Amp 3 for 30 min, and Amp4 for 15 min. There were 2 washes between each amplification, and all amplifications were performed at 40°C in the EZ Hybridization oven. Slides were mounted in Prolong Gold Anti-fade reagent (Invitrogen). For chromogen staining, the RNAscope 2.5 HD Duplex Reagent Kit (ACD 320701) was used. The sections were treated with H 2 O 2 for 10 min and then incubated with the protease K IV for 30 min. After hybridization with probes, the sections were washed 2 times, followed with amplification from Amp1 to Amp6 with 2 washes in between. To detect the red signal component, Red-B was diluted 1:60 in component Red-A and incubated on the tissue for 10 minutes at room temperature. Slides were rinsed two times in water to stop the chromogen reaction. Amplification continued with Amp7 through Amp10, followed by detection of the green signal, which was achieved by diluting component Green-B 1:50 in component Green-A and incubating for 10 minutes at room temperature. Counterstaining was performed by immersing the slides in 50% hematoxylin for 30 seconds. The slides were then dried and mounted in VectaMount mounting medium (Vector Laboratories).

Fos Studies and Colocalization Counts Franklin and Paxinos (2008) Franklin K.B.J.

Paxinos G. The Mouse Brain in Stereotaxic Coordinates, Compact Third Edition. Glp1r-ires-Cre; Ai3-EYFP, Glp1r-ires-Cre; Ai6-EYFP, Mc4r-T2a-Cre; Ai3-EYFP, Mc4r-T2a-Cre; Ai6-EYFP, Oxt-ires-Cre; Ai3-EYFP, Oxt-ires-Cre; Ai6-EYFP, Crh-ires-Cre; Ai3-EYFP and Crh-ires-Cre; Ai6-EYFP and wildtype animals were used for these analyses. Brains were sectioned at 40 um into 4 series. Each group (genotype and condition) had tissue from 3 independent animals. Two series from each animal spanning the entire PVH were used for quantification analyses (∼8 sections per series from -0.58 to -1.22 mm to Bregma). GFP and Oxytocin or GFP and Fos colocalization counts were performed using the Cell Counter feature on ImageJ (v. 1.48). DAB images were aligned to PVH regional boundaries from “The Mouse Brain in Stereotaxic Coordinates” by. Detailed experimental procedures for Fos studies are provided below. Fasted versus Refed Condition All animals used for these analyses were males to exclude any Fos induction due to cycling. Mice were socially isolated for at least 1 week to acclimate for social stress. Mice were handled daily and euthanized in their homecages to assuage experimenter-related stress responses. Additionally, all mice had ad libitum water access and nesting material for the duration of the experiment to control for Fos induction due to thirst or thermogenesis. All animals were food-deprived for 24 hours. Fasted mice were then euthanized in this state near the beginning of the light cycle. Refed mice were given access to chow pellets for 2 or 8 hours near the beginning of the light cycle and were euthanized immediately following this refeeding period. Ad libitum food access mice were euthanized near the beginning of the light cycle.

Slice Whole-Cell Electrophysiology Male mice of age 5-9 weeks were either fasted overnight, or fasted overnight and allowed to feed for 2 hours prior to decapitation under isoflurane anesthesia. Brains were rapidly removed and placed in ice-cold sucrose artificial cerebrospinal fluid (ACSF): (in mM) 194 sucrose, 20 NaCl, 4.4 KCl, 2 CaCl2, 1 MgCl2, 1.2 NaH2PO4, 10.0 glucose, and 26.0 NaHCO3 saturated with 95% O2/5% CO2. Three hundred micron slices were prepared using a Leica VT1000 vibratome (Wetzlar, Germany). Brain slices containing the PVH were obtained and stored at approximately 30°C for an hour in a heated, oxygenated holding chamber containing artificial cerebrospinal fluid (ACSF) (in mmol/L) 124NaCl, 4.4 KCl, 2 CaCl2, 1.2 MgSO4, 1 NaH2PO4, 10.0 glucose, and 26.0 sodium bicarbonate. They were then transferred to a submerged recording chamber maintained at approximately 30°C (Warner Instruments, Hamden, Connecticut) for around 30 min before recording. Recording electrodes (3–5 MΩ) were pulled with a Flaming-Brown Micropipette Puller (Sutter Instruments, Novato, CA), using thin-walled borosilicate glass capillaries. During inhibitory transmission experiments, recording electrodes were filled with (in mmol/L) 135 K+-gluconate, 5 NaCl, 2 MgCl 2 , 10 HEPES, 0.6 EGTA, 4 ATP, 0.4 GTP, pH 7.4, 290 to 295 mOsmol. All experiments were conducted under the current clamp configuration. For recording basal properties of PVHGlp1r neurons, Glp1r-ire-Cre mice received bilateral viral injection of AAV5-EF1α-DIO-eYFP in the PVH allowing identification of PVHGlp1r neurons for electrophysiological recordings. Signals were acquired via a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, California), digitized at 20 kHz, filtered at 3 kHz, and analyzed using Clampfit 10.2 software (Molecular Devices). The position of each cell was mapped to brain schematics from the “Paxinos and Franklin’s the Mouse in Stereotaxic Coordinates” atlas. Experiments in which changes in access resistance were greater than 20% before and after the experiments were not included in the data analysis. For recording CNO-mediated effects of PVHGlp1r neurons, Glp1r-ire-Cre mice received bilateral viral injection of either pAAV8-hSyn-DIO-hM3D(Gq)-mCherry or pAAV8-hSyn-DIO-hM4D(Gi)-mCherry in the PVH allowing identification of PVHGlp1r neurons for electrophysiological recordings. CNO was washed on at 10μM to demonstrate the functionality of DREADD.

Viral Injections Krashes et al., 2011 Krashes M.J.

Koda S.

Ye C.

Rogan S.C.

Adams A.C.

Cusher D.S.

Maratos-Flier E.

Roth B.L.

Lowell B.B. Rapid, reversible activation of AgRP neurons drives feeding behavior in mice. −1 and the pipette was withdrawn 5 min after injection. For electrophysiology experiments, AAV5-EF1a-DIO-EYFP-WPRE-pA, pAAV8-hSyn-DIO-hM3D(Gq)-mCherry or pAAV8-hSyn-DIO-hM4D(Gi)-mCherry was bilaterally injected into the PVH (25-50 nl, bregma: AP: −0.70 mm, DV: −4.70 mm, L: +/−0.20 mm). For fiber photometry experiments, AAV1-CAG-FLEX-GCaMP6s-WPRE-SV40 was unilaterally injected into the PVH (25-50 nl, bregma: AP: −0.70 mm, DV: −4.70 mm, L: +/−0.20 mm). For chemogenetic experiments, pAAV8-hSyn-DIO-hM3D(Gq)-mCherry, pAAV8-hSyn-DIO-hM4D(Gi)-mCherry or pAAV8-hSyn-dF-HA-KORD-IRES-mCitrine was bilaterally injected into the PVH (25-50 nl, bregma: AP: −0.70 mm, DV: −4.70 mm, L: +/−0.20 mm). For chronic neural perturbation experiments AAV-DJ-CMV-DIO-eGFP-2A-TeNT or AAV5-EF1a-DIO-EYFP-WPRE-pA was bilaterally injected into the PVH (25-50 nl, bregma: AP: −0.70 mm, DV: −4.70 mm, L: +/−0.20 mm). Stereotaxic injections were performed as previously described () Mice were anaesthetized with isoflurane and placed into a stereotaxic apparatus (Stoelting Just for Mice). For postoperative care, mice were injected intraperitoneally with meloxicam (0.5 mg per kg). 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 S58 Stimulator) was used to control injection speed at 25 nl minand the pipette was withdrawn 5 min after injection. For electrophysiology experiments, AAV5-EF1a-DIO-EYFP-WPRE-pA, pAAV8-hSyn-DIO-hM3D(Gq)-mCherry or pAAV8-hSyn-DIO-hM4D(Gi)-mCherry was bilaterally injected into the PVH (25-50 nl, bregma: AP: −0.70 mm, DV: −4.70 mm, L: +/−0.20 mm). For fiber photometry experiments, AAV1-CAG-FLEX-GCaMP6s-WPRE-SV40 was unilaterally injected into the PVH (25-50 nl, bregma: AP: −0.70 mm, DV: −4.70 mm, L: +/−0.20 mm). For chemogenetic experiments, pAAV8-hSyn-DIO-hM3D(Gq)-mCherry, pAAV8-hSyn-DIO-hM4D(Gi)-mCherry or pAAV8-hSyn-dF-HA-KORD-IRES-mCitrine was bilaterally injected into the PVH (25-50 nl, bregma: AP: −0.70 mm, DV: −4.70 mm, L: +/−0.20 mm). For chronic neural perturbation experiments AAV-DJ-CMV-DIO-eGFP-2A-TeNT or AAV5-EF1a-DIO-EYFP-WPRE-pA was bilaterally injected into the PVH (25-50 nl, bregma: AP: −0.70 mm, DV: −4.70 mm, L: +/−0.20 mm).

Optic Fiber Implantation For fiber photometry experiments, optical fibers (fiber: core = 400 μm; 0.48 NA; M3 thread titanium receptacle; Doric Lenses) were implanted unilaterally over the PVH (25-50 nl, bregma: AP: −0.80 mm, DV: −4.60/-4.65 mm, L: +/−0.20 mm). Fibers were fixed to the skull using C&B Metabond Quick Cement and dental acrylic and mice were allowed 2 weeks for recovery before acclimatization investigator handling (12 h light/dark cycle starting at 6am) for 1 week before experiments.

In Vivo Fiber Photometry Recordings started 4-6 weeks after cranial surgeries to allow for adequate recovery and viral expression. All recordings were done in the home cage of the individually-housed experimental animal near the beginning of the light cycle. Mice were allowed to adapt to the tethered patchcord for 2 days prior to experiments (core = 400 μm; 0.48 NA; M3 connector; Doric Lenses) and given 10 minutes to acclimate to the tethered patchcord prior to any recording. Continuous <20 μW blue LED at 470 nm and UV LED at 405nm served as excitation light sources, driven by a multichannel hub (Thorlabs), modulated at 211hz and 511hz respectively, and delivered to a filtered minicube (FMC5, Doric Lenses) before connecting through optic fibers to a rotary joint (FRJ 1 × 1, Doric Lenses) to allow for movement GCaMP6s calcium GFP signals and UV autofluorescent signals were collected through the same fibers back to the dichroic ports of the minicube into a femtowatt silicon photoreceiver (2151, Newport). Digital signals were then demodulated, amplified, and collected through a lock-in amplifier (RZ5P, Tucker-Davis Technologies). Data was collected through the software Synapse (TDT), exported via Browser (TDT), and analyzed in Microsoft Excel. Synchronized multi-angled high definition videos were recorded for time-locked data analysis in Synapse. 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 FCS4) 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 4-6 weeks prior to experiments, which allows GCaMP6s expression to stabilize. Chow/Object/HFD Presentation A 10-minute baseline recording was performed in fasted (overnight food-deprived; 16 hr) or fed (ad libitum food access) mice before a 1) chow pellet, 2) non-salient, non-edible object (cap of 15 mL Falcon tube) or 3) pellet (60 kcal% fat HFD; Research Diets D12492i) was introduced into the animal’s cage and the response was recorded for a minimum of 5 minutes after presentation. Importantly, all animals were acclimated to chow pellets, the non-salient, non-edible object and HFD pellets prior to the recordings to eliminate novelty responses. Each animal was tested for the response to food or object in both the fasted and fed conditions. Inaccessible Food A 10-minute baseline recording was performed in fasted (overnight food-deprived; 16 hr) mice before a caged chow pellet enclosed in a Teaball (Yinggesi Premium Stainless-Steel Tea Filter) was introduced into the cage. This specific presentation allows for the sensory detection of food through visual and olfactory cues but restricts consumption. After 20 minutes, the chow pellet was uncaged from the Teaball and the animals were given access to the food inside for another 20 minutes. Importantly, all animals were acclimated to chow pellets and the Teaball prior to the recordings to eliminate novelty responses. Ghrelin/Saline Injections A 10-minute baseline recording was performed in fed (ad libitum food access) mice before an intraperitoneal injection of saline (200 uL) or ghrelin (60 ug/mouse using PBS as vehicle at a total volume of 200 uL) was administered. Neural activity was recorded for another 10 minutes and food intake was assessed at the conclusion of the experiment to ensure the orexigenic properties of ghrelin were intact. Photometry Analysis Data were down sampled to 8Hz and signals were normalized against the mean of 5 min baseline (F0) before stimulus presentation (time = 0) to calculate Fn = F(t)/F0. Quantified data were compared using ΔF = (F(t)-F0)/F0 of 5 min window means before and after time 0. Additional analyses were performed for 1) the Glp1r HFD fed condition experiment ( Figure S5 G) and 2) the Mc4r chow fasted condition experiment ( Figure S5 M) whereby quantification comparison was made using 2 min windows immediately before and after time 0, HFD vs object and chow vs object, respectively.

Body Composition Analysis Taicher et al., 2003 Taicher G.Z.

Tinsley F.C.

Reiderman A.

Heiman M.L. Quantitative magnetic resonance (QMR) method for bone and whole-body-composition analysis. Measures of fat and lean body mass were determined in live mice using quantitative magnetic resonance (QMR) spectroscopy (EchoMRI 3-in-1, Echo MRI) (). Weekly measurements from each experimental group were taken across 16 weeks.

Indirect Calorimetry Garfield et al., 2015 Garfield A.S.

Li C.

Madara J.C.

Shah B.P.

Webber E.

Steger J.S.

Campbell J.N.

Gavrilova O.

Lee C.E.

Olson D.P.

et al. A neural basis for melanocortin-4 receptor-regulated appetite. Krashes et al., 2011 Krashes M.J.

Koda S.

Ye C.

Rogan S.C.

Adams A.C.

Cusher D.S.

Maratos-Flier E.

Roth B.L.

Lowell B.B. Rapid, reversible activation of AgRP neurons drives feeding behavior in mice. Energy expenditure was assessed by measuring oxygen consumption as previously described () using a comprehensive lab animal monitoring system (CLAMS; Columbus Instruments). Briefly, food was removed near the beginning of the light cycle and animals were injected with saline or CNO (1 mg/kg) in a crossover design and data was collected over the entire light cycle. Food was returned at the onset of the dark cycle.

Chemogenetic Food Intake Studies Garfield et al., 2015 Garfield A.S.

Li C.

Madara J.C.

Shah B.P.

Webber E.

Steger J.S.

Campbell J.N.

Gavrilova O.

Lee C.E.

Olson D.P.

et al. A neural basis for melanocortin-4 receptor-regulated appetite. Krashes et al., 2011 Krashes M.J.

Koda S.

Ye C.

Rogan S.C.

Adams A.C.

Cusher D.S.

Maratos-Flier E.

Roth B.L.

Lowell B.B. Rapid, reversible activation of AgRP neurons drives feeding behavior in mice. Food intake studies on chow were performed as previously described () All animals were singly housed for at least 2.5 weeks following surgery and handled for 10 consecutive days before the assay to reduce stress response. Studies were conducted in a homecage environment with ad libitum food access. A full trial consisted of assessing food intake from the study subjects after they received injections of saline or CNO (0.3 mg/kg) in a crossover design across the course of 3 days to ensure the effects of CNO-mediated stimulation had dissipated. Mice with ‘missed’ injections 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. Dark-cycle hM3Dq feeding studies were conducted between 6:00pm to 9:00pm and food intake was monitored 0.5 h, 1 h, 2 h and 3 h after i.p. injection. For post-fast refeed hM3Dq studies, animals were fasted overnight at 5:00pm and food returned the following morning at 9:00am. Food intake was monitored 0.5 h, 1 h, 2 h and 3 h after i.p. injection. Light-cycle hM4Di feeding studies were conducted in calorically replete ad libitum fed mice between 9:00am to 12:00pm and food intake was monitored 0.5 h, 1 h, 2 h and 3 h after i.p. injection. Nguyen et al., 2016 Nguyen K.P.

O'Neal T.J.

Bolonduro O.A.

White E.

Kravitz A.V. Feeding Experimentation Device (FED): a flexible open-source device for measuring feeding behavior. Burmeister et al., 2017 Burmeister M.A.

Ayala J.E.

Smouse H.

Landivar-Rocha A.

Brown J.D.

Drucker D.J.

Stoffers D.A.

Sandoval D.A.

Seeley R.J.

Ayala J.E. The hypothalamic glucagon-like peptide 1 receptor is sufficient but not necessary for the regulation of energy balance and glucose homeostasis in mice. Sisley et al., 2014 Sisley S.

Gutierrez-Aguilar R.

Scott M.

D'Alessio D.A.

Sandoval D.A.

Seeley R.J. Neuronal GLP1R mediates liraglutide's anorectic but not glucose-lowering effect. Mice injected with KORD were housed in custom-built cages equipped with a feeding experimental device (FED) for accurate, real-time measurements of food intake (), an infrared light-penetrable shelter, a water bottle and a ceiling mounted camera to allow for animal tracking throughout the arena. Designated FED and Home Zones were drawn around the FED and shelters, respectively using Noldus Tracking Software. All experiments were conducted during the light cycle between 9:00am and 10:00am. Each individual animal was tested under 5 independent conditions in a crossover design: 1) ad lib fed administered the vehicle DMSO, 2) ad lib fed administered the KORD ligand SALB, 3) ad lib fed administered SALB pre-treated with liraglutide, 4) fasted administered DMSO and 5) fasted administered DMSO pre-treated with liraglutide. SALB (10 mg/kg) or DMSO were administered subcutaneously. Lira (200 ug/kg BW) was delivered subcutaneously 1 hour before the initiation of the experiment. Peripheral doses were chosen based on previously demonstrated anorectic efficacy (). Food intake was automatically registered by the FED and confirmed via recorded video. All behavior was recorded and scored using EthoVision XT 10 Software (Noldus).

Progressive Ratio Task Fully acclimated and handled mice were maintained at 85% of their respective body weights during the training protocol in the testing chamber (Noldus Phenotyper) where familiarity of the pellet delivery (active versus non-active nosepoke) and food retrieval systems (pellet dispenser into dish) were obtained. Training sessions took place over 5 consecutive days on a fixed ratio 1 (FR1) schedule. To continue training, each animal had to consume at least 5 pellets (20 mg grain pellets; TestDiet) during each two-hour session. After 5 training sessions, animals that were not nosepoking sufficiently to earn at least 5 pellets were eliminated from the study. For PVHGlp1r neuron silencing experiments, breakpoint testing was performed on a progressive ratio 2 (PR2) schedule, such that each successive food pellet increased the nosepoke schedule by two additional responses. Breakpoint was defined as the maximum number of consecutive nosepokes an animal performed to procure a single food pellet. Mice received either an intraperitoneal saline or CNO (0.3 mg/kg) injection immediately before being placed in the test chamber.

Corticosterone Assay All experiments were performed in ad libitum fed animals near the beginning of the light cycle. Food was removed 2 hours prior to intraperitoneal injection with CNO or saline in a crossover design across the course of 3 days to ensure the effects of CNO-mediated stimulation had dissipated. One hour after injection, mice were restrained, and tail vein blood collected. Serum was analyzed for corticosterone content using a solid-phase ELISA according to the manufacturer’s protocol (IBL-America).

Open Field Assay Burnett et al., 2016 Burnett C.J.

Li C.

Webber E.

Tsaousidou E.

Xue S.Y.

Bruning J.C.

Krashes M.J. Hunger-driven motivational state competition. Experiments were performed as previously described (). Briefly, mice received injections of saline or CNO (0.3 mg/kg) in a crossover design, 10 minutes before being placed in the open field apparatus during a 10-min trial. This experiment was repeated with the opposite treatment 1 week later to limit sensitization to the chamber. The open field arena (Stoelting) measured 40x40x35cm. A square sector designated as the center zone was measured at 20x20x35cm in the center of the chamber. All trials were recorded on video using EthoVision XT 10 (Noldus).