Dopamine-inhibited POMCDrd2+ neurons in the ARC acutely regulate feeding and body temperature

Resource availability

Lead contact. Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jens C. Brüning (bruening@sf.mpg.de).

Data and code availability. RNA-Seq data of ribosome-associated mRNA (bacTRAP) data of POMC and POMCDrd2+ neurons have been deposited at NCBI Gene Expression Omnibus (GEO) and are publicly available as of the date of publication (accession GSE210311).

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Experimental model and subject details

Mouse husbandry. All animal procedures were conducted in compliance with protocols approved by the local authorities (Bezirksregierung Köln). Permission for breeding and experiments on mice was issued by the Department for Environment and Consumer Protection - Veterinary Section, Cologne, North Rhine-Westphalia, Germany [(§11) 576.1.35.2.G 07/18, 84-02.04.2017.A058]. Mice were group-housed in individually ventilated cages at 22°C–24°C with 12-hour light/12-hour dark cycle and ad libitum access to water and normal chow diet (NCD; ssniff Spezialdiäten, catalog V1554-703) containing 67 kJ% carbohydrate, 23 kJ% protein, and 10 kJ% fat. Mice had restricted access to food only for time-limited periods during glucose and body temperature measurements via infrared thermography (Teledyne FLIR, FLIR E6-XT) and immediately before organ harvest. Group-housing was randomized by weaning pups into allocated cages of 2 to 5 animals without prior knowledge of genotypes. Mice were single-housed to measure indirect calorimetry (Promethion, Sable Systems) or to determine body temperature via infrared thermography. Following these procedures, female mice were regrouped into their original cage distributions, while male mice remained single-housed.

Mouse lines. C57BL/6N mouse line was obtained from Charles River. For RNA in situ hybridization 12-week-old male mice were used.

NPYGFP [B6.FVB-Tg(Npy-hrGFP)1Lowl/J] mouse line has been described (47) and was obtained from Jackson Laboratory (stock number: 006417; RRID: IMSR_JAX:006417). NPYGFP+/– mice of both sexes were used for electrophysiological studies at age between 11 and 20 weeks.

POMCGFP [C57BL/6J-Tg(Pomc-EGFP)1Low/J] mouse line has been described (48) and was obtained from Jackson Laboratory (stock number: 009593; RRID: IMSR_JAX:009593). POMCGFP+/– mice of both sexes were used for electrophysiological studies at age 11–20 weeks.

Drd2Cre [Tg(Drd2-cre)ER44Gsat] mouse line has been described (49) (MGI: 3836635).

POMCDre mouse line has been generated in our laboratory and been described (4).

R26-lx-rx-ZsGreen (ROSA26-CAGS-lox-STOP-lox-rox-STOP-rox-ZsGreen) mouse line has been described (50).

R26-lx-rx-EGFP-L10a and R26-lx-rx-hM3Dq-ZsGreen mouse lines have been generated in our laboratory and been described (4). Mice have originally been named “ROSA26lSlrSrEGFPL10a” and “ROSA26lSlrSrhM3Dq.”

R26-rx-EGFP-L10a and R26-rx-ZsGreen mouse lines have been generated in our laboratory by crossing previously published (4, 50) R26-lx-rx-transgenic mice to mice that ubiquitously express Cre recombinase [BALB/c-Tg(CMV-cre)1Cgn/J, Jackson Laboratory stock number: 003465; RRID: IMSR_ JAX:003465] (51), which were kept in-house on a C57BL/6N background.

All above-listed mouse lines were maintained on and regularly backcrossed to a C57BL/6N background (Charles River) in the facility of the Max Planck Institute for Metabolism Research. For breeding purposes of animals, refer to Breeding strategies.

Breeding strategies. Both for male and female mice, 8 weeks was considered the minimum age for all crossings.

NPYGFP, POMCGFP, Drd2Cre, and POMCDre mice were heterozygously maintained by crossing NPYGFP+/–, POMCGFP+/–, Drd2Cre+/–, or POMCDre+/– to WT C57BL/6N animals (Charles River). NPYGFP+/– or POMCGFP+/– animals were consequently used for electrophysiological recordings, Drd2Cre+/– and POMCDre+/– mice were crossed to each other to obtain Drd2Cre+/– POMCDre+/– double-transgenic mice, which were subsequently bred with the R26-lx-rx-transgenic animals.

R26-lx-rx-ZsGreen, R26-lx-rx-EGFP-L10a, and R26-lx-rx-hM3Dq-ZsGreen were homozygously maintained. R26-lx-rx-transgenic+/+ mice were crossed to Drd2Cre+/– POMCDre+/– double-transgenic animals to obtain experimental animals of 4 possible genotypes: Drd2Cre–/– POMCDre–/– R26-lx-rx-transgenic+/–, Drd2Cre–/– POMCDre+/– R26-lx-rx-transgenic+/–, Drd2Cre+/– POMCDre–/– R26-lx-rx-transgenic+/–, or Drd2Cre+/– POMCDre+/– R26-lx-rx-transgenic+/– mice. The latter (triple-transgenic Drd2Cre+/– POMCDre+/– R26-lx-rx-transgenic+/– mice) are referred to as “POMCDre Drd2Cre R26-lx-rx-transgenic” animals throughout the manuscript. For genotype controls Drd2Cre–/– POMCDre–/– R26-lx-rx-transgenic+/–, Drd2Cre–/– POMCDre+/– R26-lx-rx-transgenic+/–, and Drd2Cre+/– POMCDre–/– R26-lx-rx-transgenic+/– have been used as indicated in the respective method description. Metabolic phenotyping of Drd2Cre–/– POMCDre+/–R26-lx-rx-transgenic+/– mice has been described in detail (4) and is not further addressed in this manuscript. Resulting POMCDre Drd2Cre R26-lx-rx-ZsGreen animals and control littermates were used for immunohistological analyses and electrophysiological recordings. Resulting POMCDre Drd2Cre R26-lx-rx-EGFP-L10a animals were used for immunohistological analyses and bacTRAP translational profiling. Resulting POMCDre Drd2Cre R26-lx-rx-hM3Dq-ZsGreen and control littermates were used for RNA in situ hybridization analyses and metabolic phenotyping.

R26-rx-EGFP-L10a and R26-rx-ZsGreen mice were homozygously maintained. R26-rx-transgenic+/+ mice were crossed to POMCDre+/– transgenic animals to obtain experimental animals of 2 genotypes: POMCDre–/– R26-rx-transgenic+/– or POMCDre+/– R26-rx-transgenic+/– mice. The latter are referred to as “POMCDre R26-rx-EGFP-L10a” or “POMCDre R26-rx-ZsGreen” animals throughout the manuscript. Resulting POMCDre R26-rx-EGFP-L10a animals were used for bacTRAP translational profiling of the whole POMC neuronal population. POMCDre R26-rx-ZsGreen mice were used for electrophysiological recordings of the whole POMC neuronal population.

Method details

RNA in situ hybridization (RNAscope). RNA in situ hybridization was performed on tissue samples of male WT C57BL/6N mice (Charles River) at 12 weeks of age and of male and female POMCDre Drd2Cre R26-lx-rx-hM3Dq-ZsGreen mice and control littermates (Drd2Cre–/– POMCDre–/– R26-lx-rx-hM3Dq-ZsGreen+/– and Drd2Cre+/– POMCDre–/– R26-lx-rx-hM3Dq-ZsGreen+/– mice) between 14 and 17 weeks of age, which were fasted at time point –120 minutes during the light cycle, i.p. injected with 3 mg/kg CNO in 0.9% saline at time point –60 minutes, and transcardially perfused at time point 0 minutes. Perfusion and tissue sectioning was performed as described above. To stain for RNA in situ, the RNAscope method by Advanced Cell Diagnostics was applied. All utilized reagents and probes can be found in Supplemental Table 1. In brief, slides were incubated at 60°C for approximately 6 hours, treated with 1× Target Retrieval Reagent (Advanced Cell Diagnostics, catalog 323100) at 99°C for 10 minutes, washed in sterile H2O for 15 seconds, washed in 100% EtOH for 3 minutes, and consequently dried overnight. The following day tissues were bordered with a hydrophobic ImmEdge Pen (Biozol, catalog VEC-H-4000) and incubated with Protease Plus (Advanced Cell Diagnostics, catalog 323100) for 25 minutes at 40°C. Samples were washed twice in sterile H2O for 2 minutes, and prewarmed (40°C) probe mix was applied to the sections. The probe mix contained up to 4 probes, which were amplified using the RNAscope 4-Plex Ancillary Kit for Multiplex Fluorescent Kit v2 (Advanced Cell Diagnostics, catalog 323120). All probes were applied at concentrations recommended by the manufacturer with following exceptions: Mm-Pomc was diluted 1:4, Mm-Agrp 1:2, Mm-Drd1a 1:1.5, Mm-Drd2 1:1.5, and Mm-Fos 1:2 respective to the recommended concentrations. RNAscope 4-plex negative (Advanced Cell Diagnostics, catalog 321831) and positive-control probes (Advanced Cell Diagnostics, catalog 321811) were processed in parallel with the target probes. Slides were incubated with the probe mix for 2 hours at 40°C and washed twice in 1× wash buffer (Advanced Cell Diagnostics, catalog 323100) for 2 minutes. All following incubation steps were performed at 40°C followed by a wash step as before: AMP1 for 30 minutes, AMP2 for 30 minutes, AMP3 for 15 minutes, HRP-C1 for 15 minutes, C1-fluorophore for 30 minutes, HRP blocker for 15 minutes, HRP-C2 for 15 minutes, C2-fluorophore for 30 minutes, HRP blocker for 15 minutes, HRP-C3 for 15 minutes, C3-fluorophore for 30 minutes, HRP blocker for 15 minutes, HRP-C4 for 15 minutes, and C4-fluorophore for 30 minutes. For fluorescent probe detection, the fluorophores Opal 520 (PerkinElmer, catalog FP1487001KT), Opal 570 (PerkinElmer, catalog FP1488001KT), Opal 620 (PerkinElmer, catalog FP1495001KT), and Opal 690 (PerkinElmer, catalog FP1497001KT) were applied at dilutions between 1:750 and 1:2,000 depending on the further shelf life of the reagent. Sections were incubated for 1 minute at room temperature with DAPI nuclear counterstain (Advanced Cell Diagnostics, catalog 323100), coverslipped in ProLong Gold Antifade Mountant (Thermo Fisher, catalog P36931), and stored in the dark at 4°C until imaged. All RNA in situ hybridizations were visualized on a confocal Leica TCS SP-8-X microscope.

Data analysis of RNA in situ hybridization (RNAscope). Microscopic images of RNA in situ hybridization were visualized using the image-processing software ImageJ (1.53f51, NIH). For counting of cells coexpressing a certain target, the Cell Counter plug-in (by Kurt De Vos, University of Sheffield, Sheffield, United Kingdom) was used. Agrp+ or Pomc+ areas defined the cell region of interest (ROI) in which coexpression of the given target was evaluated; 3 or more dots of target signal within a given cell defined it as positive and less signal as negative. For signal intensity analysis, ROIs were defined outlining the Pomc signal: in the image channel depicting Pomc signal, a 2.0 pixel median filter was applied, followed by the Triangle autothresholding method for white objects on black background. Thus defined particles were filtered for a minimum size of 20 μm and defined as ROIs including holes. Correct outlining of all POMC neurons was ensured by visual judgment. Cells that were considered wrongly outlined were excluded from the analysis. POMC neurons were furthermore divided into Drd2+ or Drd2– subpopulations by defining any cell with 3 or more dots of Drd2 signal as positive, with less signal as negative. After ROI definition of POMC neurons and their allocation into subpopulations, they were measured for integrated density and area within the given target image channel. Intensity was defined as intensity (a.u.) = raw integrated density/area. For signal intensity analysis of RNA in situ hybridization, intensities of single cells were plotted as assigned to their respective subpopulation.

Electrophysiological measurements. For electrophysiological measurements NPYGFP, POMCGFP, POMCDre Drd2Cre R26-lx-rx-ZsGreen, and POMCDre R26-rx-ZsGreen mice of both sexes were used at 11–20 weeks of age. The electrophysiological experiments were carried out essentially as described (4). In brief, the animals were lightly anesthetized with isoflurane (AbbVie; catalog B506) and decapitated, and coronal brain slices of 280 μm thickness containing the ARC were cut with a vibration microtome (Leica Biosystems; Leica VT1200) under cold (4 °C), carbogenated (95% O2 and 5% CO2), glycerol-based modified artificial cerebrospinal fluid (GaCSF) (52). GaCSF contained 244 mM glycerol, 2.5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 1.2 mM NaH2PO4, 10 mM HEPES, 21 mM NaHCO3, and 5 mM glucose, adjusted to pH 7.2 with NaOH. Afterward, slices were transferred into carbogenated aCSF at 36°C for 30–40 minutes and kept at room temperature until further usage for electrophysiological recordings. aCSF contained 125 mM NaCl, 2.5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 1.2 mM NaH2PO4, 21 mM NaHCO3, 10 mM HEPES, and 5 mM glucose, adjusted to pH 7.2 with NaOH. During electrophysiological recordings, brain slices were continuously superfused with carbogenated (95% 02; 5% CO2) aCSF at a flow rate of ~2.5 mL/min. In all recordings, the aCSF contained 10−4 M picrotoxin (Sigma-Aldrich; catalog P1675), 5 × 10–6 M CGP-54626 (Biotrend, catalog BN0597), 5 × 10−5 M DL-AP5 (Biotrend, catalog BN0086), and 10−5 M CNQX (Sigma-Aldrich, catalog C127) to block GABAergic and glutamatergic synaptic input.

Current-clamp recordings were performed at approximately 32°C in the perforated patch-clamp configuration. Neurons were visualized with a fixed-stage upright microscope (BX51WI, Olympus) using ×60 water-immersion objectives (LUMplan FL/N ×40, 0.8 numerical aperture, 2 mm working distance; LUMplan FL/N ×60, 1.0 numerical aperture, 2 mm working distance, Olympus) with fluorescence optics and infrared differential interference contrast optics (53). Neurons were identified by their anatomical location in the ARC and by their GFP or ZsGreen fluorescence that was visualized with an X-Cite 120 illumination system (EXFO Photonic Solutions) in combination with a Chroma 41001 filter set (excitation: HQ480/×40; beam splitter: Q505LP; emission: HQ535/50m). Electrodes with tip resistances between 4 and 7 MΩ were fashioned from borosilicate glass (0.86 mm inner diameter; 1.5 mm outer diameter; GB150-8P, Science Products) with a vertical pipette puller (PP-830, Narishige). All recordings were performed with an EPC10 patch-clamp amplifier (HEKA) controlled by the program PatchMaster (version 2x90; HEKA) running under Windows. In parallel, data were recorded using a micro1410 data acquisition interface and Spike 2 (version 7.01, both from CED). Current-clamp recordings were sampled at 25 kHz and low-pass-filtered at 2 kHz with a 4-pole Bessel filter. The calculated liquid junction potential of 14.6 mV between intracellular and extracellular solution was compensated (calculated with Patcher’s Power Tools plug-in from https://www3.mpibpc.mpg.de/groups/neher/index.php?page=software for Igor Pro 6; Wavemetrics).

Perforated patch experiments were conducted using protocols modified from previous studies (54, 55). Recordings were performed with a pipette solution containing 140 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 0.1 mM EGTA and 2 mM MgCl2, adjusted to pH 7.2 with KOH. ATP and GTP were omitted from the intracellular solution to prevent uncontrolled permeabilization of the cell membrane (56). The patch pipette tip was filled with internal solution and backfilled with internal solution, which contained the ionophore amphotericin B (Sigma-Aldrich; catalog A4888) to achieve perforated patch recordings (57, 58), 0.02% tetramethylrhodamine-dextran (Invitrogen, catalog D3308) to monitor the stability of the perforated membrane, and 1 % biocytin (Sigma-Aldrich; catalog B4261) to label the recorded neuron. Amphotericin B was dissolved in DMSO to a concentration of 40 μg/μL (Sigma-Aldrich; catalog D8418) following the protocols of a previous study (4). The used DMSO concentration (0.1–0.3%) had no noticeable effect on the investigated neurons. The ionophore was added to the modified pipette solution shortly before use. The final concentration of amphotericin B was approximately 120 to 160 μg/mL. Amphotericin solutions were prepared from undissolved weighted samples (stored at 4°C protected from light) every recording day. During the perforation process, access resistance (Ra) was monitored continuously, and experiments started after Ra values reached a steady state (~10–20 minutes) and the action potential amplitude was stable. To confirm the integrity of the perforated patch, Ra was monitored. A change to the whole-cell configuration was also indicated by dextran fluorescence in the cell body.

To investigate the dopamine responses in AgRP/NPY neurons of NPYGFP mice or in POMC neurons of POMCGFP mice, increasing dopamine (Sigma-Aldrich; catalog H8502) concentrations of 0.3 μM, 3 μM, 10 μM, and 30 μM were sequentially bath-applied for 10 minutes each concentration. To study the effect of Sst (Sigma-Aldrich; catalog S1763), in POMC neurons of POMCDre R26-rx-ZsGreen mice or in POMCDrd2+ neurons of POMCDre Drd2Cre R26-lx-rx-ZsGreen mice, Sst was bath-applied at a concentration of 300 nM for 5 minutes.

Data analysis was performed with Spike2 (Cambridge Electronics), GraphPad Prism (version 8.2; GraphPad Software Inc), and custom-made analysis scripts written in Igor Pro. To visualize the significance of the dopamine responses, we used z score plots. For the bar graphs in Figures 24 and Supplemental Figure 1, we used the “3 times SD” (3σ) criterion to classify a neuron as a responder on the single-cell level: a neuron was considered responsive if the change in firing frequency induced by drug application was 3 times larger than the SD. Means and respective SDs of spontaneous action potential firing were calculated from 5-minute periods (divided into 30 bins, each 10 seconds long) during baseline conditions and at the end of the drug application. In neurons that did not elicit action potentials, we used changes in membrane potential in a similar way as a response indicator.

bacTRAP. bacTRAP was performed on hypothalami of male and female POMCDre Drd2Cre R26-lx-rx-EGFP-L10a mice or male and female POMCDre R26-rx-EGFP-L10a mice. Animals were decapitated randomly fed at 11 to 13 weeks of age, brains were quickly removed, and hypothalami were dissected using a stainless steel brain matrix (World Precision Instruments). Samples were snap-frozen in liquid nitrogen and stored at –80°C until further processing. For POMCDre Drd2Cre R26-lx-rx-EGFP-L10a mice, hypothalami of 18–20 animals with balanced sex proportions were pooled per replicate for a total of 3 replicates. For POMCDre R26-rx-EGFP-L10a mice, hypothalami of 3 animals (2 males, 1 female) were pooled per replicate for a total of 3 replicates. The method for purifying translating ribosomes was performed as described by Heiman et al. (59) with minor modifications: in brief, 375 μL Protein A Dynabeads (Invitrogen, catalog 10001) per replicate were washed 3 times in wash buffer I (20 mM HEPES/pH 7.4, 5 mM MgCl2, 150 mM KCl, 1% Nonidet P-40, 0.5 mM DTT, and 100 μg/mL cycloheximide) and subsequently resuspended in 275 μL wash buffer I. An anti-GFP antibody mixture (50 μg of Heintz Lab TRAP anti-GFP 19C8 antibody, catalog Htz-GFP-19C8; RRID: AB_2716737 and 50 μg of Heintz Lab TRAP anti-GFP 19F7 antibody, catalog Htz-GFP-19F7; RRID: AB_2716736) was incubated with the beads overnight at 4°C with slow end-over-end mixing. The following day beads were washed 3 times in wash buffer II (20 mM HEPES/pH 7.4, 5 mM MgCl2, 150 mM KCl, 1% Nonidet P-40, 0.5 mM DTT, and 200 μg/mL cycloheximide) and subsequently resuspended in 200 μL wash buffer II. For lysis buffer preparation 1 tablet of cOmplete mini EDTA-free protease inhibitor cocktail (Sigma-Aldrich, catalog 11836170001) was dissolved in 10 mL 20 mM HEPES/pH 7.4, 5 mM MgCl2, 150 mM KCl, 0.5 mM DTT, 40 U/mL RNasin, and 100 μg/mL cycloheximide. Pooled hypothalami were homogenized in 1 mL lysis buffer on a rotating glass/teflon potter homogenizer (Braun Biotech, Potter S) at 4°C twice at 250 rpm and 9 times at 750 rpm. Homogenates were centrifuged in low binding microfuge tubes (Applied Biosystems, catalog AM12450) at 2,000g and 4°C for 10 minutes. Supernatants were consequently mixed on ice with Nonidet P-40 (AppliChem, catalog A1694,0250) and 1,2-diheptanoyl-sn-glycero3-phosphocholine (Avanti Polar Lipids, catalog 850306P) at a final concentration of 1% (w/v) and 30 mM, respectively; incubated for 5 minutes; and centrifuged for 10 minutes at 17,000g and 4°C. A total of 30 μL of the supernatant was snap-frozen in liquid nitrogen until RNA extraction and served as hypothalamic input sample for each respective IP in the analysis of translational profiling. The remaining supernatant was mixed on ice with 200 μL anti-GFP antibody-coated beads and incubated for 1 hour at 4°C with slow end-over-end mixing. Sample-bead complexes (IPs) were collected via magnet and washed 4 times with wash buffer III (20 mM HEPES/pH 7.4, 5 mM MgCl2, 350 mM KCl, 1% Nonidet P-40, 0.5 mM DTT, 100 μg/mL cycloheximide). RNA of hypothalamic input and IP samples was extracted using the RNeasy Micro Kit (QIAGEN, catalog 74004). In brief, samples were eluted off the beads by adding 350 μL RLT buffer and incubating for 5 minutes at room temperature. Subsequently manufacturer’s instructions were followed without alteration. RNA integrity was assessed using a 2100 Bioanalyzer (Agilent).

RNA-Seq. RNA-Seq was performed on bacTRAP samples of POMCDre Drd2Cre R26-lx-rx-EGFP-L10a or POMCDre R26-rx-EGFP-L10a mice. Preamplification was performed via Ovation RNA-Seq system (V2), using total RNA with both poly(T) and random primers for first-strand cDNA synthesis, followed by second-strand cDNA synthesis and isothermal strand displacement amplification. cDNA libraries were prepared from 1 ng cDNA input according to the Illumina Nextera XT DNA sample preparation protocol. After validation (Agilent 2200 TapeStation) and quantification (Invitrogen Qubit), transcriptome libraries of matching samples were pooled. Pools were quantified via Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection and sequenced on an Illumina HiSeq 4000 instrument with a 2 × 75 bp paired-end read length protocol.

bacTRAP RNA-Seq analysis. For RNA-Seq analysis of bacTRAP samples of POMCDre Drd2Cre R26-lx-rx-EGFP-L10a or POMCDre R26-rx-EGFP-L10a mice, we applied the community-curated nf-core/rnaseq analysis pipeline version 3.0 (60). The gene-level quantification was carried out using Salmon 1.4.0 (61) using the reference genome GRCm38. The differential gene expression analysis was performed using the DESeq2 1.28.0 (62) R package. Protein-coding genes were filtered for using the Ensembl (63) biomaRt (64) package.

Differentially expressed genes were identified by comparing POMCDrd2+-IP versus POMCDrd2+-IN (Padj ≤ 0.05) or POMC-IP versus POMC-IN. We furthermore filtered for cells enriched in POMC neuronal subclusters from a single-cell sequencing map of the hypothalamus (65). We additionally marked genes part of the neuropeptide signaling pathway GO term (66).

Reclustering single-cell RNA-Seq of Sst clusters. For the analysis of Th and Sst coexpressing neurons, we used the processed data from Campbell et al. (2) deposited at GEO (accession GSE93374). Based on the original annotation, we subsetted the data set to all cells in the 4 clusters “Agrp/Sst”, “Sst/Nts”, “Sst/Unc13c” and “Sst/Pthlh” that express Sst to obtain 807 cells. We then reprocessed this subset using the standard Seurat pipeline (see https://satijalab.org/seurat/articles/pbmc3k_tutorial.html) (67) with a small number of highly variable features, 400, and principal components, 20. For reclustering we employed the louvain clustering algorithm with a resolution of 2.2 to obtain 11 subclusters. Marker genes between these subclusters were calculated with Seurat’s FindMarker function using default parameters.

Indirect calorimetry. For indirect calorimetry, metabolic parameters of male and female POMCDre Drd2Cre R26-lx-rx-hM3Dq-ZsGreen mice and control littermates (Drd2Cre–/– POMCDre–/– R26-lx-rx-hM3Dq-ZsGreen+/– and Drd2Cre+/– POMCDre–/– R26-lx-rx-hM3Dq-ZsGreen+/– mice) between 12 and 17 weeks of age were assessed in a Promethion Sable System (Promethion, Sable Systems). One week prior to measurements, animals were single-housed for acclimatization in metabolic cages and were handled daily. On day of measurement the recording was started 1 hour before dark phase, and data were acquired over a course of 24 hours with ad libitum access to NCD and water. CNO was i.p. injected at concentration of 3 mg/kg in 0.9% saline both at beginning of measurement and after 4 hours. Raw data were analyzed using the software ExpeData v.1.9.22 and Sable Systems Macro Interpreter v2.38 (Promethion, Sable Systems) via an analysis script with 10-minute data binning.

Infrared thermography. For infrared thermography, imaging of male and female POMCDre Drd2Cre R26-lx-rx-hM3Dq-ZsGreen mice and control littermates (Drd2Cre–/– POMCDre–/– R26-lx-rx-hM3Dq-ZsGreen+/–, Drd2Cre+/– POMCDre–/– R26-lx-rx-hM3Dq-ZsGreen+/–, and Drd2Cre–/– POMCDre+/– R26-lx-rx-hM3Dq-ZsGreen+/– mice) at 12–20 weeks of age was performed using an FLIR E6-XT camera (Teledyne FLIR, FLIR E6-XT) equipped with a biconvex zinc selenide close-up lens for focal distances of 10.16 cm (EPSYS invent, custom made). The thermography camera was operating with a thermal sensitivity of <0.06°C, an accuracy of ±2% or ±2°C, and an infrared resolution of 240 × 180 pixels. Starting 1 week prior to measurements, mice were handled daily and acclimatized to imaging procedure. Two days prior to measurements, fur was removed from the animals in the area overlying the BAT. Mice were single-housed without nesting and minimum bedding 1 hour and fasted immediately before beginning of thermal imaging. Ad libitum access to water was maintained.

CNO was i.p. injected at concentration of 3 mg/kg in 0.9% saline and thermal images acquired at time points 0, 1, 2, 3, 4, 6, and 8 hours after CNO injection. A total of 2–7 images were acquired and analyzed of BAT and tail of freely behaving mice. For thermal imaging of the eye, mice were shortly fixed in the neck to ensure optimal focal distance. Thermal images were analyzed using the software FLIR tools V.6.4.18039.1003 by assessing the maximum temperature within eye and BAT, while defining the average temperature in a size-defined spot measurement at the tail base.

Additional methods details are in Supplemental Methods.

Statistics

For statistical analysis of electrophysiological experiments, please refer to Electrophysiological measurements. For analysis of bacTRAP data, please refer to bacTRAP RNA-Seq analysis.

All other data were statistically analyzed using the GraphPad Prism v.9.2.0 software. Given normal distribution and equal variance between groups, data was analyzed using 2-tailed Student’s t test or 2-way ANOVA/2-way mixed effects models with Holm-Šídák or Tukey’s post hoc multiple comparisons test. Pairing and repeated measurements were assigned where applicable. Data without normal distribution or equal variance were analyzed with 2-tailed Wilcoxon or Mann-Whitney rank-sum tests. Pairing was assigned where applicable.

If not stated otherwise, data are represented as mean ± SEM; individual replicates are shown as dot plots. P values less than 0.05 were considered statistically significant.

Study approval

All animal procedures were conducted in compliance with protocols approved by the local authorities (Bezirksregierung Köln, Cologne, Germany). Permission for breeding and experiments on mice was issued by the Department for Environment and Consumer Protection - Veterinary Section, Cologne, North Rhine-Westphalia, Germany [(§11) 576.1.35.2.G 07/18, 84-02.04.2017.A058].

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