Research Resource: Gene Profiling of G Protein–Coupled Receptors in the Arcuate Nucleus of the Female

Abstract

The hypothalamic arcuate nucleus controls many critical homeostatic functions including energy homeostasis, reproduction, and motivated behavior. Although G protein–coupled receptors (GPCRs) are involved in the regulation of these functions, relatively few of the GPCRs have been identified specifically within the arcuate nucleus. Here, using TaqMan low-density arrays we quantified the mRNA expression of nonolfactory GPCRs in mouse arcuate nucleus. An unprecedented number of GPCRs (total of 292) were found to be expressed, of which 183 were known and 109 were orphan GPCRs. The known GPCR genes expressed were classified into several functional clusters including hormone/neurotransmitter, growth factor, angiogenesis and vasoactivity, inflammation and immune system, and lipid messenger receptors. The plethora of orphan genes expressed in the arcuate nucleus were classified into 5 structure-related classes including class A (rhodopsin-like), class B (adhesion), class C (other GPCRs), nonsignaling 7-transmembrane chemokine-binding proteins, and other 7-transmembrane proteins. Therefore, for the first time, we provide a quantitative estimate of the numerous GPCRs expressed in the hypothalamic arcuate nucleus. Finally, as proof of principle, we documented the expression and function of one of these receptor genes, the glucagon-like peptide 1 receptor (Glp1r), which was highly expressed in the arcuate nucleus. Single-cell RT-PCR revealed that Glp1r mRNA was localized in proopiomelanocortin neurons, and using whole-cell recording we found that the glucagon-like peptide 1-selective agonist exendin-4 robustly excited proopiomelanocortin neurons. Thus, the quantitative GPCR data emphasize the complexity of the hypothalamic arcuate nucleus and furthermore provide a valuable resource for future neuroendocrine/endocrine-related experiments.

The hypothalamic arcuate nucleus is a small but complex center that controls many critical physiological functions including energy homeostasis, reproduction, locomotor activity, and motivated behaviors (1–9). Many of these functions are controlled or modulated via G protein–coupled receptors (GPCRs), such as opioid, melanocortin, neuropeptide Y (NPY), GPR54, γ-aminobutyric acid (GABA) B, serotonin, and adrenergic receptors (10–18). Although much is known about the anatomical distribution and physiological functions of many of the GPCRs that are expressed in the hypothalamus (19–22), there is still an information gap concerning the quantitative expression of GPCR subtypes and the expression of orphan GPCRs within the arcuate nucleus.

GPCRs comprise the largest family of cell surface signaling molecules and are encoded by approximately 800 genes in the human genome (23). Approximately half of these are olfactory or taste receptors, and the remaining (∼375) are nonolfactory (23). In addition, there are many orphan GPCRs, so called because the endogenous ligand and functions are not known (21, 23).

To elucidate the expression of nonolfactory GPCRs, we performed TaqMan low-density arrays (384-well setting) and investigated nonolfactory GPCR mRNA expression in the arcuate nucleus of female mice. Interestingly, we found that 292 of the 353 GPCRs in the array, of which 109 were orphans, were expressed in the arcuate nucleus. The expression of these orphans within the arcuate nucleus argues for a role in homeostatic functions, which may help to deorphanize the receptors because many neuropeptides and other ligands for GPCRs for this brain region are known (8, 21).

The array revealed that GABA_B R1 and R2 are highly expressed, which confirms our previous in vitro electrophysiology findings dating back over 20 years (18, 24–27). The array also revealed new candidates such as glucagon-like peptide (GLP) 1 receptor (Glp1r), which was highly expressed in the arcuate nucleus. As proof of principle, the GLP1-selective agonist exendin-4 induced an inward current via activation of a nonselective cationic conductance along with inhibition of an inwardly rectifying potassium conductance in proopiomelanocortin (POMC) neurons. Therefore, this quantitative analysis provides insight into the complexity of GPCR signaling in the hypothalamic arcuate nucleus and is a valuable resource for directing future experiments.

Materials and Methods

Animals and treatments

All animal treatments were in accordance with institutional guidelines based on National Institutes of Health standards and were performed with institutional animal care and use committee approval at the Oregon Health and Science University. Adult female and male wild type (C57BL/6) and POMC-enhanced green fluorescent protein (EGFP) mice (C57BL/6; originally obtained from Dr Malcolm Low) (12) were used for a GPCR low-density array and/or verification experiments. The mice were selectively bred in-house and were maintained under controlled temperature (25°C) and photoperiod (12:12 hours light/dark cycle) conditions with food and water ad libitum. To limit the influence of reproductive hormones on gene expression, adult females were ovariectomized (OVX) under ketamine-xylazine (1 and 0.1 mg/10 g, respectively) anesthesia, treated once with carprofen (4 mg/kg) for analgesia, and allowed to recover for 7 to 13 days. Gonadally intact male and female POMC-EGFP mice were used for whole-cell recording and for POMC cell harvesting.

Experimental design

RNA preparation and TaqMan GPCR low-density array analysis

OVX female POMC-EGFP mice (3–6 months old) were used for arcuate nucleus microdissection and RNA extraction. To obtain sufficient RNA for the GPCR array determinations, RNA from 6 to 7 mice was used to form 1 pool, and 4 different pools of RNA (Arc1, Arc2, Arc3, and Arc4) were prepared for the experiments. Each pool of RNA was DNase treated and reverse transcribed as described below. Initially, each pool of cDNA was subjected to quality control, which included performing real-time quantitative PCR (qPCR) analysis for the μ-opioid GPCR and the orphan GPCR12 using SYBR Green real-time PCR assays (see below). The housekeeping gene, β-actin mRNA, was also measured as an overall control gene. GPCR12, in addition to the μ-opioid receptor (28), was used for test purposes because we had found previously that this orphan receptor was expressed in the arcuate nucleus.

Samples from each pool were run as a separate GPCR array, ie, 4 different cDNA pools for 4 different arrays as outlined below. The TaqMan-type quantitative GPCR low-density array (384-well; Applied Biosystems [ABI]) was preformatted, and targets included retinal receptors, small molecule receptors, chemokine receptors, and others including classic endogenous control targets from already known and orphan GPCRs for a total of 353 GPCRs.

Tissue dissection, RNA extraction, and cDNA synthesis

The arcuate nucleus was microdissected from OVX POMC-EGFP mice, and RNA was extracted as described previously (29). In brief, a brain slicer (EM Corporation) was used to produce in each animal a 1-mm coronal block, which extended from the rostral to the caudal borders of the arcuate nucleus. This tissue block was placed in RNAlater (Ambion; Thermo Fisher Scientific Inc) for tissue RNA preservation. After 2 to 3 hours in the RNAlater solution, the arcuate nucleus was dissected with the aid of a dissecting microscope. Total RNA was extracted under RNase-free conditions using an Ambion RNAqueous Micro kit and quantified with a NanoDrop ND-100 spectrophotometer (NanoDrop Technologies). Total RNA was DNAse-1 treated (DNA-free kit; Ambion) at 37°C for 30 minutes to eliminate genomic DNA contamination. All RNA samples produced purity values with an OD_260/280 of 1.9 to 2.1. We have established the method of RNA extraction using RNAlater to protect the RNA before extraction, followed by use of an RNAqueous Micro kit for extraction to produce RNA for both the microarray and qPCR. The RNA integrity was analyzed by running 250 ng of RNA on a 1.2% denaturing agarose gel in 1× 3-(N-morpholino)propanesulfonic acid (MOPS) buffer and ribosomal RNAs 28S and 18S were visualized with ethidium bromide (Figure 1A). Reverse transcription was performed for first-strand cDNA synthesis with 400 ng of total RNA, random primers (100 ng/reaction; Promega), and SuperScript III RT (200 U/reaction; Invitrogen) in a total volume of 20 μL. Reverse transcription was conducted with the following protocol: 25°C for 5 minutes, 50°C for 60 minutes, and 70°C for 15 minutes, followed by cooling to 12°C. The cDNAs from several reactions were combined to generate each pool, and the pools were stored at −20°C. Aliquots of the respective arcuate tissue RNA were used as negative controls (no RT) and processed simultaneously with the experimental samples.

Figure 1.

Assessment of RNA quality in the samples used for PCR array analysis. A, Samples containing 250 ng of RNA from arcuate pools 1 to 4 were run on a 1.2% denaturing agarose gel in 1× MOPS buffer and visualized with ethidium bromide. The gel depicts 2 distinct ribosomal RNA bands (28S and 18S) for each sample. B and C, μ-Opioid receptor (μ-opioid R) and GPR12 mRNA levels were measured in the 4 independent RNA samples from arcuate nuclei using reverse transcription and real-time qPCR. This was done to ascertain that there were no significant differences in RNA expression measured in the 4 independent samples to be used for PCR array determinations.

qPCR of arcuate tissue cDNA using mouse GPCR low-density array

The cDNA from each arcuate pool was first evaluated using individual real-time qPCR assays for the μ-opioid receptor and GPR12. Primers were designed for GPR12 using accession number NM_00101094 (127-bp product, forward primer 445–466 nt, and reverse primer 550–571 nt). Primers for the μ-opioid receptor and the control gene (β-actin) were as described previously (30). Next, the respective cDNA pools were analyzed using the mouse TaqMan GPCR low-density array (384-well setting) according to the manufacturer's instruction on an ABI HT 7900 real-time PCR system. In brief, the samples were loaded into 8 reservoirs, each with 100 μl for a total of 800 μl for each array. A single array was run per animal pool. Four arrays were run for 4 distinct animal pools with the following reaction conditions: 50°C for 2 minutes and 94.5°C for 10 minutes, followed by 40 cycles of 97°C for 30 seconds and 59.7°C for 1 minute. In addition to the 353 GPCR genes, the GPCR array had 25 non-GPCR genes and 3 control genes: 18S, β-actin (Actb), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Verification of GPCR low-density array results using individual GPCR TaqMan assays

The results of the low-density arrays were verified using individual TaqMan qPCR assays with the same primers and probes as those used in the array and purchased individually from ABI: GABA_B receptor 2 (Gabbr2), μ-opioid receptor (Oprm1), 5-hydroxytryptamine (serotonin) (5HT) receptor 2C (Htr2c), CRH receptor 2 (Crhr2), and the control housekeeping gene β-actin (Actb). For these test experiments, we used the same arcuate tissue samples as used in the low-density arrays. The individual real-time qPCR assays were run under the following conditions: 50°C for 2 minutes, 95°C for 10 minutes, and then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute using TaqMan Gene Expression Master Mix (ABI) in 96-well plates in a 7500 fast real-time PCR system (ABI). The results were compared with the low-density array results for the same genes. In addition, the expression of selected GPCRs (GLP1R and GLP2R, μ-opioid receptor, and κ-opioid receptor) was confirmed using Power SYBR Green Master Mix (ABI) on an ABI fast real-time PCR instrument as described previously (29, 31). Primers were designed to span introns using Clone Manager software (Professional Suite version 8; Scientific and Educational Software) and synthesized by Invitrogen (Thermo Fisher Scientific Inc). Real-time qPCR primers for μ-opioid receptor and κ-opioid receptors were as described previously (30). qPCR primers for GLP1R and GLP2R, respectively, were as follows: accession number NM_021332 (109-bp product, forward primer 379–400 nt, and reverse primer 468–487 nt) and accession number NM_175681 (94-bp product, forward primer 1379–1399 nt, and reverse primer 1452–1472 nt.

POMC-EGFP cell harvest, cDNA synthesis, and single-cell PCR

These procedures were conducted as described previously (32–34). In brief, four 250-μm basal hypothalamic slices were cut on a vibratome and placed in an auxiliary chamber containing oxygenated artificial cerebrospinal fluid (aCSF). The slices were allowed to recover for 1 to 2 hours in the auxiliary chamber. Thereafter, the arcuate nucleus was microdissected and digested with protease (1 mg/ml aCSF) for approximately 15 minutes at 37°C. The cells were dispersed onto a 60-mm glass-bottom Petri dish and were perfused continually with aCSF at a rate of 2.0 ml/min. The dispersed fluorescent cells were visualized using a Leica inverted fluorescent microscope, patched, and then harvested with gentle suction to the pipette and expelled into siliconized microcentrifuge tubes containing 5 μl of RNasin solution. Individual POMC-EGFP cells were harvested from 6 animals (3 males and 3 females). Perfused aCSF was regularly collected along with the harvested cells and processed together with the cells and other control samples.

Single POMC cells were reverse transcribed for the first-strand cDNA synthesis using both random primers (100 ng/reaction) and anchored oligo(dT) primers (400 ng/reaction; Invitrogen) and SuperScript III RT (100 U/reaction) in a total volume of 20 μl. One cell from each animal was processed without the RT enzyme for a negative control. Basal medial hypothalamic tissue RNA used as positive and negative controls was processed similarly with and without RT, respectively. Initially, all GFP harvested cells were identified as POMC cells using single-cell PCR as described previously (33).

In addition, primers were designed for the GLP1R and GLP2R for single-cell PCR analysis using Clone Manager software and synthesized by Invitrogen. Single-cell GLP1R and GLP2R primers were, respectively, as follows: accession number NM_021332 (149-bp product, forward primer 474–494 nt, and reverse primer 602–622 nt) and accession number NM_175681 (171-bp product, forward primer 665–676 nt, and reverse primer 816–835 nt). POMC primers for single-cell analysis were as described previously (33).

GPCR array data analysis

The array data analysis was conducted using ABI RQ Manager 1.2 software. The cycle threshold (C_T) was set at 0.3 ΔRn (according to the manufacturer's specification) for all 4 plates, and the upper limit of the PCR cycles was 35 for a real product. Then the raw data were exported into the Microsoft Excel program for further analysis. Relative quantification analysis was done using the comparative ΔΔC_T method (35, 36). The data are expressed as an n-fold change in gene expression normalized to a reference gene, β-actin (GAPDH gave similar results, whereas 18S rRNA was expressed in quantities that were too high to provide a useful control) and relative to a calibrator (the mean ΔC_T of all GPCR genes with C_T ≤35 cycles). Means ± SEM from the 4 pools were used to determine the relative quantitative expression of GPCR mRNAs in the arcuate nucleus.

Whole-cell recording

Based on our findings that Glp1r mRNA was equally expressed in male and female POMC neurons and that the GLP1R agonist exendin-4 (200 nM; Tocris Bioscience) induced similar inward currents in male and female POMC neurons, adult male POMC-EGFP mice were used for the quantitative analysis of the effects of exendin-4 (33). Coronal slices containing the arcuate nucleus were prepared for electrophysiological recording. The electrophysiological instrumentation, recording methods, and solutions were as we have reported previously (33). Exendin-4 (200 nM) was applied to POMC neurons after the establishment of the whole-cell patch-clamp configuration. Depolarization and neuronal firing were measured under current clamp conditions. In addition, exendin-4–induced currents were measured under voltage-clamp conditions. The current-voltage relationship of the exendin-4–activated current was constructed from the currents induced by a family of voltage steps from −120 to −40 mV with a duration of 1 second (V_hold = −60 mV).

Results

Quality control of the experimental procedure

To assess the purity of the RNA samples, we measured the OD_260/280 and found this to be 2.07 ± 0.01 (n = 4), which indicates excellent purity of the extracted RNA (37). In addition, we analyzed the 28S/18S rRNA bands and found 2 distinct bands with 28S being more robust than 18S in all 4 of the pooled arcuate samples, an indication that the RNA quality was good (Figure 1A). Further quality control assays for RNA extraction and cDNA synthesis were performed through measurements of the μ-opioid receptor and GPR12 as target genes and β-actin as internal control using RT-PCR in the four different arcuate samples. Each arcuate sample was analyzed at least 6 times. This analysis revealed that the mRNA expression of the μ-opioid receptor and GPR12 were consistent across the four arcuate samples (Figure 1, B and C), suggesting that the RNA and cDNA amount and quality were similar in the 4 different arcuate samples (P > .05; one-way ANOVA).

GPCR array analysis

With use of the GPCR low-density array approach, transcript levels for 353 target GPCRs were profiled in the adult mouse arcuate nucleus on 4 different plates. Based on this analysis, we identified the expression levels of 183 of the already known GPCRs (51.84%) and 109 putative orphan GPCRs (30.88%) (Figure 2 and Tables 1–9). The remaining 61 GPCR genes in the array (17.28%), including 36 known and 25 orphan GPCRs, were undetectable (C_T >35 cycles) in the arcuate nucleus. The relative mRNA expression levels of the various GPCRs were quantified and arbitrarily classified as high-expressing (>5-fold), moderate-expressing (1- to 5-fold), or low-expressing (<1-fold) genes. The expression profiles for the 4 independent arcuate tissue pools run on the 4 different plates were consistent. Therefore, most of the 353 GPCR genes were expressed in the hypothalamic arcuate nucleus.

Figure 2.

Pie graph of classified GPCR genes (292) expressed in mouse hypothalamic arcuate nucleus. Thirteen classified groups including 8 known functional groups and 5 orphan groups of GPCR genes are represented by a different color in each group. The known functional GPCR groups contain 183 genes including neurotransmitter receptors (44; 15%), neuropeptide receptors (26; 9%), hormone receptors (31; 11%), receptors having angiogenesis and vasoactivity (10; 3%), receptors with inflammation and immune system regulation (16; 5%), lipid messenger receptors (20; 7%), growth factor receptors (12; 4%), and other known G protein-coupled receptors (24; 8%). The orphan GPCR groups contain 109 genes including class A orphans (66; 23%), class B orphans (23; 8%), class C orphans (4; 1%), nonsignaling 7TM chemokine-binding proteins (2; 1%), and other 7TM protein genes (14; 5%). The number on each slice of pie indicates the number of the genes in each classified group (see the names of the genes in each group in Tables 1–9).

Table 1.

Classification and Relative Quantity of Neurotransmitter Receptors in the Arcuate Nucleus (44)

Classification Detector	Mean ± SEM	Annotation
Acetylcholine receptors (muscarinic) (5)
Chrm1-Mm00432509_s1	10.0196 ± 1.0778	Cholinergic receptor, muscarinic 1
Chrm2-Mm01701855_s1	0.9426 ± 0.0615	Cholinergic receptor, muscarinic 2, cardiac
Chrm3-Mm00446300_s1	4.4877 ± 0.3574	Cholinergic receptor, muscarinic 3, cardiac
Chrm4-Mm00432514_s1	0.9044 ± 0.0365	Cholinergic receptor, muscarinic 4
Chrm5-Mm01701883_s1	7.4446 ± 0.3187	Cholinergic receptor, muscarinic 5
Adenosine receptors (4)
Adora1-Mm01308023_m1	14.2690 ± 4.5901	Adenosine A1 receptor
Adora2a-Mm00802075_m1	0.2879 ± 0.0255	Adenosine A2a receptor
Adora2b-Mm00839292_m1	1.7697 ± 0.0568	Adenosine A2b receptor
Adora3-Mm00802076_m1	0.2163 ± 0.0206	Adenosine A3 receptor
Adrenergic receptors (9)
Adra1a-Mm00442668_m1	6.6485 ± 0.3875	Adrenergic receptor, α1a
Adra1b-Mm00431685_m1	2.5781 ± 0.3029	Adrenergic receptor, α1b
Adra1d-Mm01328600_m1	0.1371 ± 0.0199	Adrenergic receptor, α1d
Adra2a-Mm00845383_s1	11.0508 ± 1.3903	Adrenergic receptor, α2a
Adra2b-Mm00477390_s1	0.0880 ± 0.0207	Adrenergic receptor, α2b
Adra2c-Mm00431686_s1	0.9547 ± 0.0725	Adrenergic receptor, α2c
Adrb1-Mm00431701_s1	1.9190 ± 0.0825	Adrenergic receptor, β1
Adrb2-Mm02524224_s1	0.1308 ± 0.0270	Adrenergic receptor, β2
Adrb3-Mm00442669_m1	0.0591 ± 0.0068	Adrenergic receptor, β3
Cannabinoid receptors (2)
Cnr1-Mm00432621_s1	26.9570 ± 0.4546	Cannabinoid receptor 1 (brain)
Cnr2-Mm02620087_s1	0.0306 ± 0.0091	Cannabinoid receptor 2 (macrophage)
Dopamine receptors (4)
Drd1a-Mm02620146_s1	0.6679 ± 0.0648	Dopamine receptor D1A
Drd2-Mm00438541_m1	3.1332 ± 0.1559	Dopamine receptor D2
Drd3-Mm00432887_m1	0.5728 ± 0.0507	Dopamine receptor D3
Drd5-Mm00658653_s1	0.1003 ± 0.0160	Dopamine receptor D5
GABAB receptors (2)
Gabbr1-Mm00444578_m1	171.931 ± 21.1734	γ-Aminobutyric acid (GABAB) receptor 1
Gabbr2-Mm01352561_m1	72.9596 ± 13.7708	γ-Aminobutyric acid (GABAB) receptor 2
Metabotropic glutamate receptors (6)
Grm1-Mm00810219_m1	3.7511 ± 1.0295	Glutamate receptor, metabotropic 1
Grm3-Mm00725298_m1	9.9115 ± 1.5151	Glutamate receptor, metabotropic 3
Grm4-Mm01306128_m1	1.8876 ± 0.1222	Glutamate receptor, metabotropic 4
Grm5-Mm00690332_m1	27.2579 ± 0.8546	Glutamate receptor, metabotropic 5
Grm7-Mm01189424_m1	7.0332 ± 2.2733	Glutamate receptor, metabotropic 7
Grm8-Mm00433840_m1	2.4910 ± 0.3229	Glutamate receptor, metabotropic 8
Serotonin (5-hydroxytryptamine) receptors (12)
Htr1a-Mm00434106_s1	2.1189 ± 0.1757	5-Hydroxytryptamine (serotonin) receptor 1A
Htr1b-Mm00439377_s1	14.4398 ± 0.3809	5-Hydroxytryptamine (serotonin) receptor 1B
Htr1d-Mm00434115_s1	1.1242 ± 0.2572	5-Hydroxytryptamine (serotonin) receptor 1D
Htr1f-Mm02619863_s1	0.0847 ± 0.0126	5-Hydroxytryptamine (serotonin) receptor 1F
Htr2a-Mm00555764_m1	7.7976 ± 0.3493	5-Hydroxytryptamine (serotonin) receptor 2A
Htr2b-Mm00434123_m1	0.0208 ± 0.0071	5-Hydroxytryptamine (serotonin) receptor 2B
Htr2c-Mm00434127_m1	2.0104 ± 0.2314	5-Hydroxytryptamine (serotonin) receptor 2C
Htr4-Mm00434129_m1	2.7883 ± 0.4173	5 Hydroxytryptamine (serotonin) receptor 4
Htr5a-Mm00434132_m1	4.1376 ± 0.7950	5-Hydroxytryptamine (serotonin) receptor 5A
Htr5b-Mm00439389_m1	0.7102 ± 0.1409	5-Hydroxytryptamine (serotonin) receptor 5B
Htr6-Mm00445320_m1	1.0866 ± 0.0790	5-Hydroxytryptamine (serotonin) receptor 6
Htr7-Mm00434133_m1	1.8060 ± 0.1417	5-Hydroxytryptamine (serotonin) receptor 7

Table 2.

Classification and Relative Quantity of Neuropeptide Receptors in the Arcuate Nucleus (26)

Classification Detector	Mean ± SEM	Annotation
Galanin receptors (3)
Galr1-Mm00433515_m1	1.3932 ± 0.1538	Galanin receptor 1
Galr2-Mm00726392_s1	0.4943 ± 0.0593	Galanin receptor 2
Galr3-Mm00443617_m1	0.4117 ± 0.0274	Galanin receptor 3
Bradykinin receptors (2)
Bdkrb1-Mm00432059_s1	0.2190 ± 0.0112	Bradykinin receptor B1
Bdkrb2-Mm00437788_s1	0.2436 ± 0.0214	Bradykinin receptor, β2
Kisspeptin receptor (1)
Kiss1r-Mm00475046_m1	1.6223 ± 0.1173	Kisspeptin receptor, KISS1 receptor
Neuromedin B receptor (1)
Nmbr-Mm00435147_m1	1.0793 ± 0.0780	Neuromedin B receptor
Neuromedin U receptors (2)
Nmur1-Mm00515885_m1	0.0857 ± 0.0158	Neuromedin U receptor 1
Nmur2-Mm00600704_m1	2.2792 ± 0.1854	Neuromedin U receptor 2
Neuropeptide FF/neuropeptide AF receptors (2)
Npffr1-Mm01176033_m1	2.6919 ± 0.3299	Neuropeptide FF receptor 1
Npffr2-Mm00500040_m1	0.8695 ± 0.0396	Neuropeptide FF receptor 2
Neuropeptide W/neuropeptide B receptors (1)
Npbwr1-Mm02621088_s1	0.1830 ± 0.0137	Neuropeptide B/W receptor 1
Neuropeptide Y receptors (3)
Npy1r-Mm00650798_g1	4.5690 ± 0.7657	Neuropeptide Y receptor Y1
Npy2r-Mm01956783_s1	1.8491 ± 0.0785	Neuropeptide Y receptor Y2
Npy5r-Mm02620267_s1	0.1625 ± 0.0201	Neuropeptide Y receptor Y5
Neurotensin receptors (2)
Ntsr1-Mm00444459_m1	4.4989 ± 0.3538	Neurotensin receptor 1
Ntsr2-Mm00435426_m1	39.7503 ± 4.6948	Neurotensin receptor 2
Opioid receptors (4)
Oprm1-Mm01188089_m1	7.0238 ± 0.0742	Opioid receptor, μ1
Oprd1-Mm00443063_m1	1.5361 ± 0.2264	Opioid receptor, δ1
Oprk1-Mm01230885_m1	2.1007 ± 0.4578	Opioid receptor, κ1
Oprl1-Mm00440563_m1	17.7796 ± 1.4257	Opioid receptor-like 1
Orexin receptors (2)
Hcrtr1-Mm01185778_m1	1.1484 ± 0.1203	Hypocretin (orexin) receptor 1
Hcrtr2-Mm01179312_m1	1.9816 ± 0.0953	Hypocretin (orexin) receptor 2
Tachykinin receptors (3)
Tacr1-Mm00436892_m1	2.6346 ± 0.3096	Tachykinin receptor 1
Tacr2-Mm00436898_m1	0.0667 ± 0.0139	Tachykinin receptor 2
Tacr3-Mm00445346_m1	5.1109 ± 0.5237	Tachykinin receptor 3

Table 3.

Classification and Relative Quantity of Hormone Receptors in the Arcuate Nucleus (31)

Classification Detector	Mean ± SEM	Annotation
Calcitonin receptors (2)
Calcr-Mm00432271_m1	19.5005 ± 3.0788	Calcitonin receptor
Calcr1-Mm00516986_m1	1.3965 ± 0.1857	Calcitonin receptor-like
Cholecystokinin receptors (2)
Cckar-Mm00438060_m1	2.0683 ± 0.1362	Cholecystokinin A receptor
Cckbr-Mm00432329_m1	6.0393 ± 0.7506	Cholecystokinin B receptor
Corticotropin-releasing factor receptors (2)
Crhr1-Mm00432670_m1	0.8884 ± 0.1048	Corticotropin-releasing factor receptor 1
Crhr2-Mm00438303_m1	0.0211 ± 0.0074	Corticotropin-releasing factor receptor 2
Estrogen (G protein–coupled) receptor (1)
Gpr30-Mm02620446_s1	0.2393 ± 0.0171	GPCR 30
Ghrelin receptor (1)
Ghsr-Mm00616415_m1	2.8523 ± 0.2406	Ghrelin receptor (growth hormone secretagogue receptor)
Glucagon receptor family (4)
Ghrhr-Mm01326479_m1	0.0087 ± 0.0019	Growth hormone–releasing hormone receptor
Gipr-Mm01316351_g1	0.0991 ± 0.0039	Gastric inhibitory polypeptide receptor
Glp1r-Mm00445292_m1	7.0520 ± 0.3008	Glucagon-like peptide 1 receptor
Glp2r-Mm00558835_m1	0.0328 ± 0.0063	Glucagon-like peptide 2 receptor
Gonadotrophin-releasing hormone receptors (1)
Gnrhr-Mm00439143_m1	0.0169 ± 0.0040	Gonadotrophin-releasing hormone receptor
Luteinizing hormone/choriogonadotropin receptor (1)
Lhcgr-Mm00442931_m1	0.2576 ± 0.0518	Luteinizing hormone/choriogonadotropin receptor
Melanin-concentrating hormone receptor (1)
Mchr1-Mm00653044_m1	0.7887 ± 0.0747	Melanin-concentrating hormone receptor 1
Melanocortin receptors (4)
Mc1r-Mm00434851_s1	0.0397 ± 0.0092	Melanocortin 1 receptor
Mc3r-Mm00434876_s1	7.7624 ± 0.2813	Melanocortin 3 receptor
Mc4r-Mm00457483_s1	0.9801 ± 0.0467	Melanocortin 4 receptor
Mc5r-Mm00442970_m1	0.1284 ± 0.0118	Melanocortin 5 receptor
Parathyroid hormone receptor (2)
Pthr1-Mm00441046_m1	0.6688 ± 0.1087	Parathyroid hormone receptor 1
Pthr2-Mm00653029_m1	0.7876 ± 0.1297	Parathyroid hormone receptor 2
Prokineticin receptors (2)
Prokr1-Mm00517546_m1	0.6927 ± 0.0960	Prokineticin receptor 1
Prokr2-Mm00769571_m1	1.4569 ± 0.2388	Prokineticin receptor 2
Prolactin-releasing peptide receptor (1)
Prlhr-Mm01266991_s1	0.6843 ± 0.0669	Prolactin-releasing peptide receptor
Somatostatin receptors (5)
Sstr1-Mm00436679_s1	11.1765 ± 1.4531	Somatostatin receptor 1
Sstr2-Mm00436685_g1	0.1265 ± 0.0432	Somatostatin receptor 2
Sstr3-Mm00436695_s1	4.4996 ± 0.3993	Somatostatin receptor 3
Sstr4-Mm00436710_s1	4.2868 ± 0.3161	Somatostatin receptor 4
Sstr5-Mm01307775_s1	0.9652 ± 0.0484	Somatostatin receptor 5
TSH receptor (1)
Tshr-Mm00442027_m1	3.0282 ± 0.4828	TSH receptor
TRH receptor (1)
Trhr-Mm00443262_m1	2.2997 ± 0.1972	TRH receptor

Table 4.

Classification and Relative Quantity of Angiogenesis and Vasoactive Receptors in the Arcuate Nucleus (10)

Classification Detector	Mean ± SEM	Annotation
Angiotensin receptors (3)
Agtr1a-Mm01957722_s1	0.1780 ± 0.0168	Angiotensin II receptor, type 1a
Agtr2-Mm01341373_m1	0.2825 ± 0.0271	Angiotensin II receptor, type 2
Agtrl1-Mm00442191_s1	0.2595 ± 0.0522	Angiotensin receptor-like 1
Endothelin receptors (2)
Ednra-Mm01243722_m1	1.4502 ± 0.1071	Endothelin receptor type A
Ednrb-Mm00432989_m1	21.9352 ± 2.9980	Endothelin receptor type B
Vasoactive intestinal peptide– and pituitary adenylate cyclase–activating polypeptide receptors (3)
Adcyap1r1-Mm00431683_m1	131.2449 ± 8.9925	Adenylate cyclase activating polypeptide 1 receptor 1
Vipr1-Mm00449214_m1	0.0971 ± 0.0154	Vasoactive intestinal peptide receptor 1
Vipr2-Mm00437316_m1	0.6042 ± 0.0708	Vasoactive intestinal peptide receptor 2
Vasopressin and oxytocin receptors (2)
Avpr1a-Mm00444092_m1	1.6037 ± 0.0892	Arginine vasopressin receptor 1A
Oxtr-Mm01182684_m1	1.4708 ± 0.1439	Oxytocin receptor

Table 5.

Classification and Relative Quantity of Inflammatory and Immune System Receptors in the Arcuate Nucleus (16)

Classification Detector	Mean ± SEM	Annotation
Anaphylatoxin receptors (2)
C3ar1-Mm02620006_s1	0.2775 ± 0.0535	Complement component 3a receptor 1
C5ar1-Mm00500292_s1	0.3223 ± 0.0492	Complement component 5a receptor 1
Chemokine receptors (11)
Ccr1-Mm00438260_s1	0.0413 ± 0.0098	Chemokine (C-C motif) receptor 1
Ccr2-Mm99999051_gH	0.5748 ± 0.0536	Chemokine (C-C motif) receptor 2
Ccr3-Mm00515543_s1	0.0130 ± 0.0050	Chemokine (C-C motif) receptor 3
Ccr5-Mm01963251_s1	0.1934 ± 0.0798	Chemokine (C-C motif) receptor 5
Ccr7-Mm01301785_m1	0.0083 ± 0.0013	Chemokine (C-C motif) receptor 7
Ccr9-Mm02620030_s1	0.2877 ± 0.0258	Chemokine (C-C motif) receptor 9
Cxcr3-Mm99999054_s1	0.0795 ± 0.0145	Chemokine (C-X-C motif) receptor 3
Cxcr4-Mm99999055_m1	0.1071 ± 0.1026	Chemokine (C-X-C motif) receptor 4
Cxcr6-Mm02620517_s1	0.2566 ± 0.0138	Chemokine (C-X-C motif) receptor 6
Cx3cr1-Mm02620111_s1	6.9515 ± 0.1138	Chemokine (C-X3-C) receptor 1
Xcr1-Mm00442206_s1	0.0177 ± 0.0026	Chemokine (C motif) receptor 1
Histamine receptors (3)
Hrh1-Mm00434002_s1	8.4888 ± 0.5633	Histamine receptor H1
Hrh2-Mm00434009_s1	0.0709 ± 0.0132	Histamine receptor H2
Hrh3-Mm00446706_m1	18.1531 ± 1.8979	Histamine receptor H3

Table 6.

Classification and Relative Quantity of Lipid Messenger Receptors in the Arcuate Nucleus (20)

Classification Detector	Mean ± SEM	Annotation
Leukotriene receptors (4)
Ltb4r1-Mm02619879_s1	0.0321 ± 0.0091	Leukotriene B4 receptor 1
Ltb4r2-Mm00498491_s1	0.1165 ± 0.0073	Leukotriene B4 receptor 2
Cysltr1-Mm02620326_s1	0.0753 ± 0.0124	Cysteinyl leukotriene receptor 1
Cysltr2-Mm02620584_s1	0.0866 ± 0.0142	Cysteinyl leukotriene receptor 2
Lysophospholipid (LPA) receptors (4)
Edg2-Mm00439145_m1	7.1370 ± 1.2251	LPA1, endothelial differentiation, lysophosphatidic acid GPRC 2
Edg4-Mm00469562_m1	0.9551 ± 0.0425	LPA2, endothelial differentiation, lysophosphatidic acid GPRC, 4
Edg7-Mm00469694_m1	0.1421 ± 0.0261	LPA3, endothelial differentiation, lysophosphatidic acid GPRC, 7
P2ry5-Mm00613058_s1	2.6972 ± 0.3282	LPA6, purinergic receptor P2Y, GPRC, 5
Lysophospholipid (S1P) receptors (4)
Edg1-Mm02619656_s1	2.8238 ± 1.1109	S1P1, endothelial differentiation sphingolipid GPRC, 1
Edg3-Mm02620181_s1	0.1047 ± 0.0077	S1P3, endothelial differentiation, sphingolipid GPRC, 3
Edg6-Mm00468695_s1	0.4419 ± 0.0976	S1P4, endothelial differentiation, GPRC, 6
Edg8-Mm02620565_s1	0.4196 ± 0.0341	S1P5, endothelial differentiation, sphingolipid GPRC, 8
Prostanoid receptors (8)
Ptgdr-Mm00436050_m1	0.0608 ± 0.0176	Prostaglandin D receptor
Gpr44-Mm00438315_s1	0.0730 ± 0.0108	DP2, GPCR 44
Ptger1-Mm00443098_g1	0.2651 ± 0.0285	Prostaglandin E receptor1 (subtype EP1)
Ptger2-Mm00436051_m1	0.3236 ± 0.0433	Prostaglandin E receptor2 (subtype EP2)
Ptger3-Mm01316856_m1	0.8174 ± 0.0324	Prostaglandin E receptor3 (subtype EP3)
Ptger4-Mm00436053_m1	0.7047 ± 0.0704	Prostaglandin E receptor4 (subtype EP4)
Ptgfr-Mm00436055_m1	0.1216 ± 0.0274	Prostaglandin F receptor
Tbxa2r-Mm00436917_m1	0.0674 ± 0.0068	Thromboxane A2 receptor

Table 7.

Classification and Relative Quantity of Growth Factor Receptors in the Arcuate Nucleus (12)

Classification Detector	Mean ± SEM	Annotation
Frizzled receptors (12)
Fzd1-Mm00445405_s1	2.2868 ± 0.1984	Frizzled homolog 1 (Drosophila)
Fzd2-Mm02524776_s1	0.4988 ± 0.0380	Frizzled homolog 2 (Drosophila)
Fzd3-Mm00445423_m1	19.0983 ± 1.9531	Frizzled homolog 3 (Drosophila)
Fzd4-Mm00433382_m1	3.0181 ± 0.4399	Frizzled homolog 4 (Drosophila)
Fzd5-Mm00445623_s1	13.4249 ± 0.5211	Frizzled homolog 5 (Drosophila)
Fzd6-Mm00433387_m1	3.2283 ± 0.2427	Frizzled homolog 6 (Drosophila)
Fzd7-Mm00433409_s1	2.0544 ± 0.1326	Frizzled homolog 7 (Drosophila)
Fzd8-Mm00433419_s1	1.2603 ± 0.1365	Frizzled homolog 8 (Drosophila)
Fzd9-Mm02621136_s1	0.6752 ± 0.0861	Frizzled homolog 9 (Drosophila)
Fzd10-Mm00558396_s1	2.0051 ± 0.1161	Frizzled homolog 10 (Drosophila)
Smo-Mm01162710_m1	6.7230 ± 0.3111	Smoothened homolog (Drosophila)
Frzb-Mm00441378_m1	6.4396 ± 1.0771	Frizzled-related protein

Table 8.

Classification and Relative Quantity of Other G Protein-Coupled Receptors in the Arcuate Nucleus (24)

Classification Detector	Mean ± SEM	Annotation
Bile acid receptor (1)
Gpbar1-Mm00558112_s1	0.2721 ± 0.0230	GPCR bile acid receptor 1
Calcium-sensing receptors (1)
Casr-Mm00443375_m1	0.7649 ± 0.1186	Calcium-sensing receptor
Formylpeptide receptors (2)
Fpr1-Mm00442803_s1	0.2505 ± 0.0353	Formyl peptide receptor 1
Fpr-rs2-Mm00484464_s1	0.0297 ± 0.0034	Formyl peptide receptor, related sequence 2
Hydroxycarboxylic acid (HCA) receptors (1)
Gpr81-Mm00558586_s1	0.0663 ± 0.0140	HCA1, GPCR 81
Melatonin receptors (1)
Mtnr1a-Mm00434999_m1	0.1422 ± 0.0330	Melatonin receptor 1A
Opsins and rhodopsin (1)
Rho-Mm00520345_m1	0.1942 ± 0.0267	Rhodopsin, a GPCR, extremely sensitive to light
Others (1)
Crcp-Mm01197736_m	16.5122 ± 0.1912	Calcitonin gene-related peptide-receptor component protein
Pancreatic polypeptide receptor (1)
Ppyr1-Mm00435894_s1	0.3744 ± 0.0595	Pancreatic polypeptide receptor 1
Platelet-activating factor receptor (1)
Ptafr-Mm02621061_m1	0.5617 ± 0.0502	Platelet-activating factor receptor
Protease-activated receptors (3)
F2r-Mm00438851_m1	4.1429 ± 0.2484	Protease-activated receptor 1, coagulation factor II (thrombin) receptor
F2rl1-Mm00433160_m1	0.0650 ± 0.0139	Protease-activated receptor 2, coagulation factor II (thrombin) receptor-like 1
F2rl2-Mm00438852_m1	1.5260 ± 0.2496	Protease-activated receptor 3, coagulation factor II (thrombin) receptor-like 2
Purinergic (P2Y) receptors (7)
P2ry1-Mm00435471_m1	0.2795 ± 0.0263	Purinergic receptor P2Y, G protein coupled 1
P2ry2-Mm02619978_s1	0.0587 ± 0.0182	Purinergic receptor P2Y, G protein coupled 2
P2ry4-Mm00445136_s1	0.0151 ± 0.0040	Pyrimidinergic receptor P2Y, G protein coupled, 4
P2ry6-Mm02620937_s1	0.1369 ± 0.0147	Pyrimidinergic receptor P2Y, G protein coupled, 6
P2ry12-Mm01950543_s1	0.0479 ± 0.0046	Purinergic receptor P2Y, G protein coupled 12
P2ry13-Mm01951265_s1	0.2298 ± 0.0156	Purinergic receptor P2Y, G protein coupled 13
P2ry14-Mm02035793_s1	0.4214 ± 0.0080	Purinergic receptor P2Y, G protein coupled, 14
Relaxin family peptide receptors (2)
Rxfp3-Mm00618735_s1	2.9747 ± 0.4442	Relaxin family peptide receptor 3
Rxfp4-Mm00731536_s1	0.0427 ± 0.0076	Relaxin family peptide receptor 4
Secretin receptor (1)
Sctr-Mm01290790_m1	0.5935 ± 0.0945	Secretin receptor
Transferrin receptors (1)
Tfrc-Mm00441941_m1	10.0531 ± 1.1201	Transferrin receptor

Table 9.

Classification and Relative Quantity of Orphan GPCR Genes in the Arcuate Nucleus (109)

Classification Detector	Mean ± SEM	Annotation
Class A orphans (66)
Ccrl2-Mm00516914_g1	0.4606 ± 0.0427	Chemokine (C-C motif) receptor-like 2
Cmklr1-Mm02619757_s1	0.2342 ± 0.0666	Chemokine-like receptor 1
Gpr1-Mm02620665_s1	0.1688 ± 0.0195	GPCR 1
Gpr3-Mm00433719_s1	0.4976 ± 0.0296	GPCR 3
Gpr4-Mm00558777_s1	1.3546 ± 0.2395	GPCR 4
Gpr6-Mm01701705_s1	2.5490 ± 0.3355	GPCR 6
Gpr12-Mm02343661_s1	0.8334 ± 0.0373	GPCR 12
AI853548, Gpr17-Mm02619401_s1	2.4195 ± 0.2651	GPCR 17, expressed sequence AI853548
Gpr18-Mm02620895_s1	0.4013 ± 0.0209	GPCR 18
Gpr19-Mm02619790_s1	0.4952 ± 0.0380	GPCR 19
Gpr20-Mm02620726_s1	0.0260 ± 0.0018	GPCR 20
Gpr21-Mm02620869_s1	0.5886 ± 0.0561	GPCR 21
Gpr22-Mm01291487_m1	2.2926 ± 0.3833	GPCR 22
Gpr23-Mm02620784_s1	1.1411 ± 0.1069	GPCR 23
N/A-Mm02621666_s1	0.7404 ± 0.0617	GPR25, GPCR 25
Gpr26-Mm01165717_m1	6.3768 ± 0.3539	GPCR 26
Gpr27-Mm01962535_s1	0.0326 ± 0.0118	GPCR 27
Gpr34-Mm02620221_s1	0.3407 ± 0.0442	GPCR 34
Gpr35-Mm01973686_s1	0.1457 ± 0.0256	GPCR 35
Gpr45-Mm00446398_s1	5.5220 ± 0.4795	GPCR 45
Gpr50-Mm00439147_m1	19.5940 ± 2.0275	GPCR 50
Gpr55-Mm02621622_s1	0.8677 ± 0.0713	GPCR 55
Gpr61-Mm00558495_s1	6.7480 ± 1.1120	GPCR 61
Gpr62-Mm02621101_s1	2.0888 ± 0.1987	GPCR 62
Gpr63-Mm00446159_s1	1.0420 ± 0.0699	GPCR 63
Gpr65-Mm02619732_s1	0.0749 ± 0.0168	GPCR 65
Gpr68-Mm00558545_s1	0.2831 ± 0.0234	GPCR 68
Gpr75-Mm00558537_s1	11.2089 ± 1.5338	GPCR 75
Gpr82-Mm00558788_s1	0.5621 ± 0.0572	GPCR 82
Gpr83-Mm00439103_m1	8.5449 ± 0.5664	GPCR 83
Gpr84-Mm02620530_s1	0.2354 ± 0.0216	GPCR 84
Gpr85-Mm00460767_s1	10.7271 ± 1.2817	GPCR 85
Gpr87-Mm00519013_s1	0.3322 ± 0.0396	GPCR 87
Gpr88-Mm02620353_s1	0.0466 ± 0.0060	GPCR 88
Gpr101-Mm01296083_m1	33.7668 ± 2.1204	GPCR 101
Gpr119-Mm00731497_s1	0.0742 ± 0.0166	GPCR 119
Gpr120-Mm00725193_m1	0.0205 ± 0.0037	GPCR 120
Gpr132-Mm02620285_s1	0.0178 ± 0.0017	GPCR 132
Gpr135-Mm00731507_s1	5.6510 ± 0.7205	GPCR 135
Gpr139-Mm01280440_m1	1.2749 ± 0.1576	GPCR 139
Gpr141-Mm00731518_s1	0.0074 ± 0.0020	GPCR 141
Gpr146-Mm01951835_s1	2.8621 ± 0.2153	GPCR 146
Gpr149-Mm00805216_m1	3.1200 ± 0.3126	GPCR 149
Gpr150-Mm02344527_s1	0.1833 ± 0.0152	GPCR 150
Gpr151-Mm00808987_s1	0.2428 ± 0.0177	GPCR 151
Gpr152-Mm02620975_s1	0.0469 ± 0.0039	GPCR 152
Gpr153-Mm00805218_m1	16.4656 ± 0.9116	GPCR 153
Gpr160-Mm02621016_s1	0.1267 ± 0.0347	GPCR 160
Gpr161-Mm01291057_m1	2.5561 ± 0.3186	GPCR 161
Gpr162-Mm00496577_m1	13.3626 ± 0.2260	GPCR 162
Gpr171-Mm02620738_s1	0.4417 ± 0.0133	GPCR 171
Gpr173-Mm02620389_s1	1.4268 ± 0.1131	GPCR 173
Gpr174-Mm01238430_m1	0.0084 ± 0.0018	GPCR 174
Gpr176-Mm01277657_m1	3.6780 ± 0.0754	GPCR 176
Admr-Mm01946034_s1	0.1446 ± 0.0251	GPR182, adrenomedullin receptor
Ebi2-Mm02620906_s1	0.7011 ± 0.0617	GPR183, Epstein-Barr virus–induced gene 2
Lgr4-Mm00554385_m1	6.6814 ± 0.1825	Leucine-rich repeat–containing GPCR 4
Lgr5-Mm00438890_m1	1.3949 ± 0.2244	Leucine-rich repeat–containing GPCR 5
Lgr6-Mm01291336_m1	3.9080 ± 0.2838	Leucine-rich repeat–containing GPCR 6
Lgr7-Mm01220214_m1	0.3115 ± 0.0512	Leucine-rich repeat–containing GPCR 7
Lgr8-Mm00446529_m1	0.2990 ± 0.0400	Leucine-rich repeat–containing GPCR 8
Mas1-Mm00434823_s1	0.1029 ± 0.0234	MAS1 oncogene, a GPCR, binds the angiotensin II metabolite angiotensin- (1–7)
Mrgpra3-Mm02620679_s1	0.0123 ± 0.0028	MAS-related GPR, member A3
Mrgpre-Mm02620836_s1	2.5059 ± 0.3675	MAS-related GPR, member E
Mrgprf-Mm02620618_s1	0.0250 ± 0.0040	MAS-related GPR, member F
Oxgr1-Mm01960674_s1	0.0936 ± 0.0094	Oxoglutarate (α-ketoglutarate) receptor 1
Class B orphans (23)
Bai1-Mm00558144_m1	52.0334 ± 2.8904	Brain-specific angiogenesis inhibitor 1, aphagocytic receptor
Bai2-Mm00557365_m1	4.9234 ± 0.5216	Brain-specific angiogenesis inhibitor 2, orphan receptor
Bai3-Mm00657451_m1	15.5840 ± 0.7051	Brain-specific angiogenesis inhibitor 3, orphan receptor
Cd97-Mm00516248_m1	1.8654 ± 0.0688	CD97 antigen, member of the epidermal growth factor (EGF)-TM7 family, new GPCR
Celsr1-Mm00464808_m1	3.1861 ± 0.4955	Cadherin EGF LAG 7-pass G-type receptor 1
Celsr2-Mm00457515_m1	1.9425 ± 0.0908	EGF, latrophilin 7TM domain–containing 1
Emr1-Mm00802529_m1	0.4630 ± 0.0233	EGF-like module–containing, mucin-like, hormone receptor–like sequence 1
Gpr56-Mm00817704_m1	52.3475 ± 1.5049	GPCR 56
Gpr64-Mm00724545_m1	0.3733 ± 0.0369	GPCR 64
Gpr97-Mm00499385_m1	0.0645 ± 0.0157	GPCR 97
Gpr111-Mm01281279_m1	0.1909 ± 0.0170	GPCR 111
Gpr114-Mm01247578_m1	0.0099 ± 0.0025	GPCR 114
Gpr115-Mm01279261_m1	0.0418 ± 0.0056	GPCR 115
Gpr116-Mm01269030_m1	3.9893 ± 0.3116	GPCR 116
Gpr123-Mm00624275_m1	42.3620 ± 4.4316	GPCR 123
Gpr124-Mm00475185_m1	1.5261 ± 0.2603	GPCR 124
Gpr125-Mm01211383_g1	5.8560 ± 0.4562	GPCR 125
Gpr133-Mm01203407_m1	0.0444 ± 0.0032	GPCR 133
Mass1-Mm00475232_m1	2.3356 ± 0.2509	GPR98, monogenic, audiogenic seizure susceptibility 1
Lphn1-Mm01247150_m1	58.5283 ± 4.2677	Latrophilin 1, a family of secretin-like GPCRs, α-latrotoxin (LTX) receptor
Lphn2-Mm01320597_m1	16.4134 ± 0.9633	Latrophilin 2, a family of secretin-like GPCRs, but not bind LTX
Lphn3-Mm01216694_m1	32.8496 ± 2.0521	Latrophilin 3, a family of secretin-like GPCRs, but not bind LTX
Class C orphans (4)
Gpr156-Mm00462845_m1	0.3794 ± 0.0648	GPCR 156
Gprc5a-Mm00724888_m1	0.0747 ± 0.0093	GPCR, family C, group 5, member A
Gprc5b-Mm00458150_m1	30.5954 ± 1.9440	GPCR, family C, group 5, member B
Gprc5c-Mm00548863_m1	0.7821 ± 0.1231	GPCR, family C, group 5, member
Non-signaling 7TM chemokine-binding proteins (2)
Ccrl1-Mm02620636_s1	0.1953 ± 0.0102	Chemokine (C-C motif) receptor-like 1
Darc-Mm02620117_s1	0.2885 ± 0.0531	Duffy blood group, chemokine receptor
Other 7TM proteins (14)
Cmkor1-Mm02619632_s1	0.0726 ± 0.0121	Chemokine orphan receptor 1
Gpr77-Mm01267981_s1	0.1266 ± 0.0151	GPCR 77
Gpr92-Mm02621109_s1	0.3266 ± 0.0374	GPCR 92
Gpr103-Mm01294559_m1	1.8488 ± 0.0771	GPCR 103
Gpr108-Mm00712856_m1	9.7793 ± 1.4049	GPCR 108
Gpr109a-Mm02620500_s1	0.0107 ± 0.0018	GPCR 109A
Gpr137-Mm01231752_g1	27.4696 ± 0.6611	GPCR 137
Gpr137b-Mm00652182_m1	11.4195 ± 1.0647	GPCR 137B
Gpr143-Mm00440553_m1	0.0241 ± 0.0046	GPCR 143
Gpr154-Mm00558817_m1	1.4863 ± 0.2508	GPCR 154
Gpr175-Mm01183739_m1	1.3931 ± 0.1338	GPCR 175
Grm2-Mm01235831_m1	1.9818 ± 0.2209	GPCR, family C, group 1, member B
Tm7sf3-Mm01255186_m1	7.5160 ± 0.2914	TM7 superfamily member 3
N/A-Mm02621695_s1	0.5950 ± 0.0693	mCG6879 Celera annotation, hypothetical protein

Overall GPCR expression profile in hypothalamic arcuate nucleus

The known GPCR genes that were expressed in the arcuate nucleus (183) were divided into 8 functional groups and the orphan GPCRs (109) into 5 orphan groups as shown in Tables 1 to 9 and in Figure 2. For the known GPCRs, these included 44 neurotransmitter receptors, 26 neuropeptide receptors, 31 hormone receptors, 10 receptors related to angiogenesis and vasoactivity, 16 receptors involved with inflammation and immune systems, 20 lipid messenger receptors, 12 growth factor receptors, and 24 other known GPCRs (Figure 2 and Tables 1 to 8). Of the 109 orphan GPCRs, 66 belong to class A (rhodopsin), 23 to class B (adhesion), 4 to class C (other GPCRs), 2 to nonsignaling 7-transmembrane (TM) chemokine-binding proteins, and 14 to other 7TM protein genes (Figure 2 and Table 9).

Verification of low-density array results using individual TaqMan GPCR assays

To verify the quantitative results of the GPCR low-density array we chose 4 GPCRs that were found to be expressed in high to low levels (GABA_B-R2 [Gabbr2], μ-opioid receptor [Oprm1], 5HT receptor 2c [Htr2c], and CRH receptor 2 [Crhr2], as well as β-actin as an internal control), to be analyzed in individual TaqMan qPCR assays. As expected, all 4 GPCR genes were expressed in the arcuate nucleus in the following order: Gabbr2 ≫ Oprm1 > Htr2c > Crhr2, which was similar to the rank order of expression of these genes as revealed by the TaqMan low-density array (R² = 0.9944; P < .005). Therefore, the relative expression of these 4 GPCRs was similar using the 2 different assay methods in 2 different PCR systems (ABI HT 7900 and ABI 7500), supporting the accuracy and reliability of the GPCR low-density array analysis.

Expression of the glucagon receptor family

The glucagon receptor family of GPCRs includes GHRH- receptor (Ghrhr), gastric inhibitory polypeptide receptor (Gipr), Glp1r, and Glp2r. Of these, Glp1r mRNA was found to be highly expressed (relative expression [RE], 7.05 ± 0.30) in the arcuate nucleus of POMC-EGFP mice, whereas the mRNA levels of the other receptors in this family were barely detectable (Figure 3 and Table 3). Glp1r vs Glp2r in the arcuate nucleus was also analyzed in wild-type C57 mice (n = 3) using qPCR (SYBR Green method) with similar results; ie, Glp1r mRNA was highly expressed (RE, 10.07 ± 0.60), whereas Glp2r mRNA could hardly be detected (RE, 0.01 ± 0.01).

Figure 3.

Glucagon receptor family mRNA expression in the arcuate nucleus. Bar graphs illustrating the relative mRNA expression of the glucagon receptor family as measured by GPCR array.

Glp1r vs Glp2r in arcuate nucleus POMC neurons

Our finding that the GLP1R was highly expressed in the arcuate nucleus is consistent with earlier observations that this receptor is involved in inhibition of feeding (38). Therefore, we measured the expression and function of GLP1R in arcuate POMC neurons. The studies revealed that Glp1r mRNA was expressed in at least 50% of POMC neurons in both males and females (Figure 4A), but this transcript could not be detected in NPY/agouti-related peptide (NPY/AgRP) neurons (data not shown). Consistent with the GPCR array measurements of low expression of Glp2r mRNA (RE, 0.03 ± 0.01) in the arcuate nucleus, Glp2r transcripts could not be detected in POMC neurons. We next examined the effects of a selective GLP1R agonist, exendin-4, on POMC neurons (39, 40). As shown in Figure 4B, exendin-4 (200 nM) induced a depolarization (5.3 ± 1.8 mV, n = 5) and increased firing in 3 of 5 cells (the other 2 cells did not respond). To examine whether exendin-4 directly activated POMC neurons, whole-cell current was measured in the presence of a synaptic blockade cocktail of drugs [tetrodotoxin, 1 μM; picrotoxin, 100 μM; and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione, 20 μM)]. As shown in Figure 4C, exendin-4 induced an inward current in 70% (n = 16) of POMC neurons with a mean amplitude of 5.2 ± 0.6 pA. The current-voltage plots revealed that the GLP1R agonist activated a nonselective cation conductance along with inhibiting an inwardly rectifying potassium conductance (Figure 4, D and E, respectively).

Figure 4.

GLP1R expression and function in arcuate POMC neurons. A, Representative gel illustrating the mRNA expression of Glp1r in arcuate POMC neurons. Thirty-six neurons were analyzed from each of 3 females and 3 males, and at least 50% of POMC neurons were positive for Glp1r mRNA. MM, molecular marker; −RT, cell reacted without RT; + and −, tissue RNA reacted with and without RT. B, Whole-cell current-clamp recording showing that the GLP1R agonist exendin-4 (200 nM) induced a depolarization and increased firing in POMC neurons. Resting membrane potential was −73 mV. C, In the voltage clamp and in the presence of synaptic blockade, exendin-4 induced an inward current. D and E, current-voltage plots revealed that the inward current was generated by the activation of a nonselective cation current (D, reversal at −40 mV) and inhibition of an inwardly rectifying K⁺ current (E, reversal at −80 mV).

Discussion

With a TaqMan low-density array assay, we investigated the mRNA expression of 353 nonolfactory GPCRs and found that most of these genes were expressed in the mouse arcuate nucleus, including 183 known and 109 orphan GPCRs. The known GPCR genes expressed were classified into 8 functional groups, including neurotransmitter/neuropeptide receptors, hormone receptors, angiogenesis/vasoactive receptors, inflammatory/immune system receptors, lipid messenger receptors, and growth factor receptors. The orphan genes expressed were grouped into 5 structure-related classes including class A (rhodopsin), class B (adhesion), class C (other GPCRs), nonsignaling 7TM chemokine-binding proteins, and other 7TM proteins. Therefore, the arcuate nucleus of the hypothalamus is richly endowed with both known and orphan GPCRs.

Based on the analysis of independent biological samples run on replicate plates, the arcuate GPCR expression profile was consistent across the different plates. Similarly, individual qPCR assays of selected genes, ranging from high to very low expression levels in the array, exhibited the same relative expression of these genes in individual real-time PCR analysis. Of the genes tested individually (Gabbr2, Oprm1, Htr2c, and Crhr2), Gabbr2 was the most highly expressed, followed by Oprm1, Htr2c, and finally Crhr2, which was barely detectable, essentially as noted in the array analysis. The very low expression of Crhr2 (RE, 0.02 ± 0.01) in the mouse arcuate nucleus is also consistent with a previous report on the expression of this receptor gene (41). Therefore, the quantitative array profile of GPCRs in the arcuate nucleus appears to be consistent and should represent a reliable measure of the expression levels of known and orphan GPCRs. Moreover, the PCR array analysis is congruent with the electrophysiological analysis of the GABA_B, μ-opioid, and 5HT receptor 2C–mediated responses in hypothalamic arcuate neurons (18, 24–27, 42).

Known GPCRs

Many of the known neurotransmitter GPCRs, which were presently identified as being moderate to highly expressed, have been documented in the arcuate nucleus, eg, adrenergic receptors α_1a, α_1b, α_2a, and β₁ and serotonergic receptors 5HT_1B, 5HT_1A, 5HT_2A, and 5HT_2C, all of whose expression and functions in the arcuate nucleus are relatively well known (43–48). Less is known about the quantitative expression of many of the other known GPCRs that were currently identified in the arcuate nucleus of adult female mice (Tables 1–8), eg, the glucagon receptor family, tachykinin receptors, RFamide receptors, calcitonin receptors, relaxin receptors, and frizzled receptors.

The arcuate nucleus of the hypothalamus is composed of multiple neuronal types that control many homeostatic functions of which the control of energy homeostasis and reproduction have been the most extensively investigated (7, 49, 50). For example, neurons expressing NPY/AgRP stimulate feeding but are also involved in the control of reproduction via modulation of GnRH neuronal excitability (51). Importantly, the α₂ adrenergic receptor appears to be involved in the NPY-induced feeding behavior and may also act in part to modulate the NPY-induced LH surge in rodents (52, 53). Several NPY receptors including Y1, Y2, and Y5 are all expressed in the arcuate nucleus (54, 55) with highest expression of Y1 (RE, 4.57 ± 0.76) and lowest expression of Y5 mRNA (RE, 0.16 ± 0.02) (Table 2). NPY neurons project to and synapse on dopamine and POMC cells in the arcuate nucleus (56, 57), an indication that NPY receptors are expressed on these neurons. The gut hormone ghrelin also stimulates feeding, and, not surprisingly, we found that the ghrelin receptor (GH secretagogue receptor) is expressed in the arcuate nucleus in relatively high quantities (RE, 2.85 ± 0.24).

Neurons expressing POMC/cocaine- and amphetamine-regulated transcript (CART) inhibit feeding and may also be involved in the control of GnRH neurons (58, 59). Although much is known about POMC peptides and their receptors, the CART receptor(s) has not yet been identified and may be among the orphan GPCRs identified in the arcuate nucleus (Table 9). The POMC peptide α-melanocyte-stimulating hormone (α-MSH), acting primarily through the melanocortin receptors 3 and 4 (MC3R and MC4R), has received much attention for its role in the control of energy homeostasis (13, 60, 61). MC4R deficiencies (mutations) lead to severe obesity in human and animal models, and MC3R mutation appears also to be associated with obesity (61, 62). Currently, we have identified 4 melanocortin receptors (MC1, MC3, MC4, and MC5) in the arcuate nucleus, of which the Mc3r transcript was by far the most highly expressed (RE, 7.76 ± 0.28) followed by much lower expression of the Mc4r transcript (RE, 0.98 ± 0.05). These results are consistent with previous reports documenting the mRNA expression of melanocortin receptors in the hypothalamus (61, 63–67). The functional significance of the low expression of Mc1r and Mc5r mRNAs within the arcuate nucleus is uncertain because these receptors are believed to be important in the periphery, eg, for skin pigmentation (MC1R) and sebaceous lipid production (MC5R) (67).

The proglucagon peptide, which is processed to glucagon in the pancreas, is processed to GLP1 and GLP2 in the brain and in the gut (for review, see ref. 68). These peptides act at the GLP1R and GLP2R, respectively (68, 69). We found that Glp1r but not Glp2r mRNA was highly expressed in the arcuate nucleus, and Glp1r mRNA (not Glp2r) was specifically identified in male and female POMC neurons. Importantly, a selective and potent agonist (exendin-4) for GLP1R (39) depolarized and increased the firing of the anorexigenic POMC neurons (Figure 4). Moreover, in the presence of synaptic blockade, the GLP1R agonist induced an inward (depolarizing) current in most POMC neurons via activation of nonselective cation conductance. Collectively, these findings suggest that functional GLP1Rs are expressed in POMC neurons, and their activation leads to increased excitation of POMC neurons. These actions of this postprandial-released gut hormone augment actions of insulin in POMC neurons (70). This could be a mechanism by which GLP1 and its agonist (exendin-4) attenuate feeding (38, 71). In comparison to Glp1r mRNA, Glp2r mRNA was expressed at many-fold lower levels in the arcuate nucleus (Figure 3). This finding is consistent with previous in situ hybridization studies, which localized Glp2r mRNA primarily outside the arcuate nucleus within the dorsomedial hypothalamus (72). In addition, intracerebroventricular injection of GLP2 induced cFos expression in the dorsomedial hypothalamus but not in the arcuate nucleus (72), a further indication that GLP2Rs may not be highly expressed or active in the arcuate nucleus. It is therefore interesting that it has been reported that Glp2r mRNA and protein are highly expressed in arcuate neurons including POMC neurons (73). The reason for the discrepancy is unclear but could be related to different animal models and experimental approaches. Although central nervous system GLP2Rs appear to be involved in the regulation of food intake in rodents, the cellular targets are not known but may involve functional interaction (cross-talk) between the GLP1- and GLP2-responsive neurons given that the anorectic effect of GLP2 was potentiated in GLP1R knockout animals (74).

GPR54, the kisspeptin 1 (KISS1) receptor, was found to be moderately expressed (RE, 1.6 ± 0.12) in the mouse arcuate nucleus (Table 2). In contrast, using a transgenic GPR54 LacZ knock-in mouse model, GPR54 mRNA was not detected in the arcuate nucleus (75). However, KISS1 has been found to excite POMC neurons in the arcuate nucleus, an indication that the kisspeptin receptor is expressed and may play a role in the control of feeding (16). Neurons expressing KISS1 are localized in the arcuate nucleus and coexpress the tachykinin, neurokinin (NK) B, as well as the opioid peptide, dynorphin (76, 77). NKB is the ligand for the tachykinin receptor 3 (Tacr3), which was one of the high-expressing GPCRs in our analysis (RE, 5.11 ± 0.52), and Tacr3 mRNA is expressed in KISS1/NKB neurons (77, 78). Both KISS1, NKB, and their respective receptors have been shown to be essential for sexual development and reproductive function in human and animal models (79–84). In addition, we found that Tacr1 (substance P receptor) mRNA was expressed in moderate levels (RE, 2.63 ± 0.31), whereas the mRNA expression of the Tacr2 (ligand, neurokinin A) was quite low (RE, 0.07 ± 0.01) in the mouse arcuate nucleus. Immunoreactive substance P has been identified in the rhesus macaque infundibular nucleus (85), and substance P mRNA is also expressed in the mouse arcuate nucleus (O. K. Rønnekleiv, unpublished observations). The current findings are consistent with a recent publication illustrating that NKB, NKA, and substance P all induced membrane depolarization in mouse arcuate KISS1 neurons (86). We found that κ-opioid receptor mRNA was moderately expressed (RE, 2.10 ± 0.46) within the arcuate nucleus. As stated above, the arcuate KISS1 neurons coexpress dynorphin, the ligand for the κ-opioid receptors. In addition, these neurons express κ-opioid receptor mRNA, and the mRNA expression levels of both dynorphin and its receptor are inhibited by estradiol (77). Interestingly, based on a series of neuroanatomical and neuroendocrine analyses, a model has been presented showing that NKB and dynorphin may act autosynaptically on arcuate KISS1 neurons to shape pulsatile secretion of GnRH (77).

We found moderate expression of the relaxin family peptide receptor 3 (Rxfp3) in the arcuate nucleus. This receptor has been identified as the receptor for the relaxin-3 (RLN-3) peptides (87). Both RLN-3 and its receptor are widely distributed in the mouse brain, and centrally administered RLN-3 induces cFos expression in different brain regions including the supraoptic nucleus and the arcuate nucleus (88–90). It is therefore, not surprising that centrally administered RLN-3 facilitates both water and food intake (88, 89). However, overall very little is known about the mechanism by which RLN-3 and its receptor induce feeding.

Calcitonin receptor (Calcr) mRNA was found to be highly expressed (RE, 19.50 ± 3.08) in the arcuate nucleus. A related peptide receptor, calcitonin receptor-like (Calcrl), was also detected, but at a much lower level. A prior immunocytochemical analysis also found a high level of immunoreactive calcitonin receptor in the rat arcuate nucleus as well as in numerous other brain regions (91). Calcitonins are a family of peptides, including calcitonin, aminoprocalcitonin, calcitonin gene–related peptide, amylin, and adrenomedullin, which can act on Calcr, Calcrl, or on heterodimers of these receptors with single transmembrane proteins (92, 93). Activation of Calcr or Calcrl affects primarily calcium homeostasis (92). In addition, the calcitonin-calcitonin receptor system may be involved in regulating energy homeostasis (93). However, the specific role of the calcitonins and their receptors in the arcuate nucleus is not known and warrants further investigation.

Of interest is that 10 growth factor receptors of the frizzled family were found to be expressed in the arcuate nucleus and the majority at quite high levels. In particular, the frizzled homolog 3 and 5 receptors were highly expressed (RE, 19.10 ± 1.95 and 13.42 ± 0.52, respectively) in this brain region in support of previous publications (94, 95). This family of receptors is known primarily to be critically involved in fetal development. However, evidence suggests that these receptors, which are activated by the Wingless-Int (WNT) family of lipoglycoproteins, are important in the adult brain and other organs (96). The WNTs comprise a large family of highly conserved growth factors that upon binding to frizzled receptors cause accumulation of β-catenin in the cytoplasm, which translocates to the nucleus and alters transcription of target genes (97). WNTs through binding to frizzled receptors can also signal via pathways involving calcium/calmodulin-dependent kinase II and protein kinase C or GTP-binding proteins, leading to activation of phospholipase C and phosphodiesterase (98). However, the function and signaling of frizzled receptors within the arcuate nucleus in adult animals appear to be unexplored.

In the low-expressing GPCR group, we identified GPR30 (RE, 0.24 ± 0.13), also known as G protein–coupled estrogen receptor 1 (GPER1) (99–102). Considerable evidence indicates that GPR30 is an estrogen receptor (ER) at the plasma membrane and signals independent of ERα and ERβ (99). GPR30 stimulates calcium oscillations and GnRH release in immature rhesus monkey GnRH neuronal cultures (103, 104); however, a physiological role for GPR30 in the arcuate nucleus of adult animals has not been identified (105, 106).

Orphan GPCRs

We arbitrarily identified these orphan genes as follows: high-expressing (>5-fold), including GPR50, GPR56, GPR75, GPR85, GPR153, and GPR brain-specific angiogenesis inhibitor (Bai) 1 and Bai3; moderate-expressing (between 1- and 5-fold), including GPR6, GPR17, GPR23, GPR176, and Bai2; and low-expressing (<1-fold), the largest group of orphan GPCRs including GPR1, GPR3, and GPR12.

GPR50, a member of the rhodopsin GPCR family, shares significant sequence homology with the two melatonin receptors (MT1 and MT2) but does not bind melatonin (107) and is, therefore, still considered an orphan GPCR. We found that this transcript is one of the high expressing genes (RE, 19.59 ± 2.03) in the arcuate nucleus. Previous in situ hybridization analysis had already revealed that GPR50 mRNA is expressed in the ventromedial hypothalamus including the arcuate nucleus (107). Immunoreactive GPR50 neurons have been identified in the dorsomedial hypothalamus but have been more difficult to detect in the arcuate nucleus (108, 109). GPR50 is believed to be involved in adaptive thermogenesis in mammals (110).

GPR75, also of the rhodopsin family of orphan GPCRs, is highly expressed (RE, 11.21 ± 1.53) in the arcuate nucleus. This receptor is a novel chemokine receptor, which is expressed in the brain, spinal cord, and in the pancreas (111, 112). Although GPR75 has limited homology (12%–16%) with chemokine receptors, it is activated by the chemokine (C-C motif) ligand 5 (CCL5; also called RANTES [regulated upon activation, normal T-cell expressed and secreted]) in pancreatic β cells, which causes intracellular calcium release and increased insulin secretion (112, 113). In the hippocampal cell line HT22, which endogenously expresses GPR75, CCL5 reduced the neurotoxicity of amyloid-β peptides by activating phospholipase C and phosphoinositide 3-kinase (113). The role of GPR75 in the arcuate nucleus has not been investigated but could be involved in anti-inflammatory responses associated with a high-fat diet (114).

GPR56, a member of the adhesion GPCR family, is one of the highest expressing (RE, 52.35 ± 1.50) orphan GPCRs in the arcuate nucleus. This transcript appears to be important for the development of the cortex because mutations in GPR56 cause bilateral frontoparietal polymicrogyria (115, 116). Possible ligands for GPR56 are collagen, type III and/or transglutaminase 2 (115–117). A role for GPR56 in the arcuate nucleus needs to be elucidated.

Bai1 to Bai3 of the adhesion GPCR family were also highly expressed in the arcuate nucleus with Bai1 ≫ Bai3 ≫ Bai2 (RE, 52.03 ± 2.89, 15.58 ± 0.71, and 4.92 ± 0.52, respectively). These receptors are involved not only in angiogenesis and phagocytosis but may also play important roles in neurogenesis, synaptogenesis, and regulation of spine morphology (118–120). Importantly, it has been found that Bai1 is enriched in postsynaptic density in mouse brain and signals through Gα_12/13 to activate the Rho pathway as shown in human embryonic kidney 293 cells (119). The role of Bai transcripts (proteins) within the arcuate nucleus needs to be investigated but could be associated with spine formation and synaptic remodeling within this important nucleus (118, 120).

In summary, this quantitative analysis of GPCR expression in the arcuate nucleus should serve as a resource on which to further elucidate the functional significance of known GPCRs as well as help to deorphanize the orphan GPCRs. The expression of these orphans within the arcuate nucleus argues for their role in important homeostatic functions, such as metabolism and energy homeostasis, as well as temperature regulation and reproduction.