Gene 553 (2014) 1–6
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The G protein-coupled receptor GPR162 is widely distributed in the CNS and highly expressed in the hypothalamus and in hedonic feeding areas Vanni Caruso, Maria G. Hägglund, Luca Badiali, Sonchita Bagchi, Sahar Roshanbin, Helgi B. Schiöth, Robert Fredriksson ⁎ Department of Neuroscience, Functional Pharmacology, Uppsala University, Husargatan 3, Uppsala 75124, Sweden
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Article history: Received 24 February 2014 Received in revised form 18 September 2014 Accepted 19 September 2014 Available online 29 September 2014 Keywords: GPCRs GPR162 Energy homeostasis Hedonic feeding
a b s t r a c t The Rhodopsin family is a class of integral membrane proteins belonging to G protein-coupled receptors (GPCRs). To date, several orphan GPCRs are still uncharacterized and in this study we present an anatomical characterization of the GPR162 protein and an attempt to describe its functional role. Our results show that GPR162 is widely expressed in GABAergic as well as other neurons within the mouse hippocampus, whereas extensive expression is observed in areas related to energy homeostasis and hedonic feeding such as hypothalamus, amygdala and ventral tegmental area, regions known to be involved in the regulation of palatable food consumption. © 2014 Elsevier B.V. All rights reserved.
1. Introduction G protein-coupled receptors (GPCRs) are a class of integral membrane proteins characterized by extracellular N-termini, intracellular C-termini and seven transmembrane α-helices, whose function is to mediate endogenous signals from the outside of the cell into cellular responses, through activation of GTP-binding proteins (G-proteins) (Fredriksson et al., 2003a). The superfamily of GPCRs is divided into ﬁve subfamilies named Rhodopsin, Glutamate, Adhesion, Frizzled/Taste 2 and Secretin, of which Rhodopsin are the largest component consisting of more than 670 human GPCRs including the olfactory receptors (Gloriam et al., 2007). Rhodopsin family is phylogenetically divided into α, β, γ, and δ groups, excluding the olfactory receptors. The α-group is the largest cluster with 101 human genes providing a substrate speciﬁcity for biogenic
Abbreviations: Cpu, Caudate putamen; Pir, piriform cortex; Tu, olfactory tubercle; Acb, accumbens nucleus; VP, ventral pallidum; LPO, lateral preoptic area; CA1-2-3, Cornu Ammonis 1-2-3; DG, dentate gyrus; ARC, arcuate hypothalamic nucleus; DM, dorsomedial; VMHDM, VMHC, VMHVL, ventral medial; LH, lateral hypothalamic nucleus; Pe, Perifornical nucleus; ACo, anterior cortical amygdaloid nucleus; BMA, basomedial; BLA, basolateral anterior; BLV, basolateral ventral amygdaloid nucleus; SNL SNR and SNC, substantia nigra lateral, reticulata and compact; VTA, ventral tegmental area; ML, medial mammillary nucleus; RMC, red magnocell nucleus; Sim, simple lobule; (4 & 5 Cb), 4th/5th cerebellar lobules; pcuf, preculminate ﬁssure; 3Cb, 3rd cerebellar lobule. ⁎ Corresponding author at: Institutionen för neurovetenskap, Uppsala biomedicinska centrum (BMC), Husargatan 3, Box 593, 751 24 Uppsala, Sweden. E-mail address: [email protected]
http://dx.doi.org/10.1016/j.gene.2014.09.042 0378-1119/© 2014 Elsevier B.V. All rights reserved.
ammine and several orphan GPCRs which are still uncharacterized (Sreedharan et al., 2011). The presence of signature motifs D/ERY/W in the second intercellular loop and NPxxY at the second trasmembrane 7 (TM7) is a structural characteristic of the Rhodopsin family (Palczewski et al., 2000). Seven GPCRs of the Rhodopsin family were previously found by searches with Hidden Markov Models in the Human Genscan dataset and BLAST searches in the Celera database (Fredriksson et al., 2003b). Phylogenetic analyses revealed nine new members of the Rhodopsin family including GPR153 and GPR162 (Gloriam et al., 2005). Phylogenetic analyses placed these orphan receptors in the α-group on a separate branch only containing these two receptors. Human GPR162 and GPR153 genes are closely related considering their sequence identity (Gloriam et al., 2005) and reﬁned phylogenetic analyses revealed that GPR153 and GPR162 shared a common ancestor gene that originated before the divergence of the teleost lineage (Sreedharan et al., 2011). GPR153 is highly expressed in the thalamus, cerebellum and arcuate nucleus and shows some similarity to serotonin receptors with 20% primary sequence amino acid identity (Gloriam et al., 2005). Functional data demonstrated its involvement in development and tumorigenesis in a variety of tissues (Lee et al., 2010). Neither GPR153 nor GPR162 has been characterized regarding ligand binding and no functional role has been ascribed to any of these. In this article we use a custom made polyclonal antibody to chart the expression of GPR162 in the mouse brain. We report GPR162 to be widely expressed in GABAergic as well as other neurons particularly in areas related to energy homeostasis and hedonic feeding such as the hypothalamus, amygdala and ventral tegmental areas.
V. Caruso et al. / Gene 553 (2014) 1–6
2. Materials and methods 2.1. Tissue collection and sectioning All animal work was approved by the Animal Care and Ethics Committee of Uppsala, Sweden and followed the guidelines of European Communities Council Directive (86/609/EEC). At 15 weeks of age, C57Bl6/J mice (Taconic M&B, Denmark) were euthanized with a 1:1 mixture of Dormitor (Medetomidine hydrochloride, 70 μg/g body weight, Orion Pharma, Finland) and Ketalar (ketamine hydrochloride, 7 μg/g body weight, Pﬁzer) for brain collection. Transcardial perfusion through the left ventricle was then performed with phosphate-buffered saline (PBS) followed by 4% formaldehyde (HistoLab, Sweden). After decapitation, the brain was stored in 4% formaldehyde overnight. Brain sections (7 μm) were obtained using a Microm 355S STS cool cut microtome, attached on Superfrost Plus slides (Menzel-Gläser, Germany), dried overnight at 37 °C, and stored at 4 °C until use. Speciﬁcally, for free ﬂoating tissue sections, the brain was washed in PBS, embedded in 4% agarose and sectioned (70 μm) on a Leica VT1000S vibratome (Leica Microsystems, Germany). Sections were then dehydrated through a series of methanol washes and stored in 100% methanol at 20 °C until further processing. For parafﬁn-embedded tissue sections, the brain was ﬁxed in zinc-formalin (Richard-Allan Scientiﬁc) for 24 h at 40 °C before dehydration and parafﬁn infusion (Tissue-Tek vacuum inﬁltration processor, Miles Scientiﬁc).
2.2. Western blot The whole brain from adult male C57B16/J mice (Taconic M&B, Denmark) was homogenized in homogenization buffer (50 mM Tris, 150 mM NaCl, 4 mM MgCl, 0.5 mM EDTA, 2% Triton X-100 and 1 mM Protease inhibitor PMSF (Sigma-Aldrich, USA) diluted in isopropanol) and protein concentrations were determined by protein assay DC (Bio-Rad, Hercules, USA). Equal amounts of protein (20 μg) were separated, together with PageRuler prestained protein ladder (Fermentas, Canada), on a Mini-Protean TGX gel (4–10%, Bio-Rad, Hercules, USA) in running buffer (0.1% SDS, 0.025 Tris base and 0.192 M glycine) by gel electrophoresis. The proteins were transferred to a Immobilon-P polyvinylidene ﬂuoride (PVDF) membrane (Millipore, Billerica, USA) in transfer buffer (0.025 Tris base, 0.192 M glycine and 20% methanol) and pre-blocked for 1 h in blocking buffer (5% non-fat dry milk (BioRAD, Hercules, USA) diluted in 1.5 M NaCl, 0.1 M Tris, 0.05% Tween-20, pH 8.0). In order to reveal epitope speciﬁcity, the membrane was cut in the middle, giving two membranes with equally loaded protein samples. One half of the membrane was hybridized with the primary antibody against GPR162 (peptide sequence: NH2-MARGGLGAEEASLRC-CONH2; diluted 1:500 in 5% non-fat dry milk, rabbit-anti-GRP162, Innovagen, Sweden). The other half of the membrane was hybridized with GPR162 primary antibody that was pre-blocked for 1 h at room temperature with excess of the same synthetic peptide that was used to generate the antibody. The hybridization was then performed overnight at 4 °C. After washes in water, the membranes were incubated for 1 h with horseradish peroxidase conjugated secondary antibody (diluted 1:10,000, goat-anti-rabbit, Invitrogen, USA) followed by detection with the enhanced chemiluminescent (ECL) method. The membranes were incubated for 3 min in a 1:1 mixture of luminol/enhancer and peroxidase buffer solutions (Immun-Star HRP, Bio-Rad, Hercules, USA) and developed on a High performance chemiluminescence ﬁlm (GE Healthcare, Waukesha, USA). In the control experiment, membranes were
Fig. 1. Characterization of the GPR162 antibody. A) Western blot revealed a speciﬁc strong band at ∼60 kDa. Peptide competition as a positive control was performed. Excess of epitope speciﬁc peptide bound GPR162 antibody up to the complete removal of the band, indicating epitope speciﬁcity of the polyclonal antibody used for our experiment. Beta actin was used as a control. B) Speciﬁcity of our antibody was further veriﬁed performing siRNA knockdown of GPR162 in the neuronal cell-line N25-2. We then compared the expression levels of GPR162 with Western blot and found that the band corresponding to GPR162 was signiﬁcantly reduced in the siRNA knockdown cells. ***P b 0.001.
hybridized overnight at 4 °C with antibodies against β-actin (dilution 1:000; Santa Cruz, CA, USA), followed by the detection procedure as above described. 2.3. Cell culture The immortalized embryonic mouse hypothalamus cell line N25/2 (mHypoE-N25/2, CellutionsBiosystems Inc., Canada) was cultured in Dulbecco's Modiﬁed Eagle Medium (DMEM [+] 4.5 g/L D-Glucose, [+] L-Glutamine, [+] Pyruvate) from Gibco, Life technologies supplemented with 50 ml fetal bovine serum (FBS) (Gibco, Life technologies), 5 ml Penicillin–Streptomycin (Pen-Strep) (Gibco, Life technologies) and 5 ml amphotericin B (Gibco, Life technologies). All cells were incubated at 37 °C with 5% CO2. 2.3.1. siRNA knockdown Cells were grown in 6 welled tissue culture plates from Falcon for 18–24 h to get 60–80% conﬂuence (Brown et al., 1978). Transfection with speciﬁc GPR162 Silencer® Select Pre-designed siRNA (Ambion Cat: 4390771) was performed using Lipofectamine® RNAiMAX reagent (Invitrogen Life Technologies) following the recommended protocol (Protocol Pub. No. MAN0007825 Rev. 1.0). Lipofectamine® RNAiMAX reagent and GPR162-siRNA (50–70 pmol) were separately diluted in Opti-MEM® Medium according to the protocol. Then the diluted
Fig. 2. Fluorescent immunohistochemistry on parafﬁn sections. Adult mouse brain sections were stained with custom-made polyclonal GPR162 antibody (red), cell nucleus marker DAPI in blue, and antibody markers in green. (A) Co-localization of GPR162 with the neuron-speciﬁc DNA-binding protein marker (NeuN) within the hippocampus on GABAergic as well as other neurons (Bregma −2.70). (B) Overlapping expression in the hypothalamus is observed after GPR162 and neuron-speciﬁc antibody NSE co-staining. (C) Further co-staining with GAD67 revealed the expression of GPR162 in GABAergic neurons in the cortex. (D) The astrocytic marker GFAP did not show overlap expression with GPR162 in the brain region between the thalamus and cerebral cortex (Bregma −2.70). (E) Epithelial cell marker Pan-Cytokeratin did not show any overlap expression with GPR162 in the hypothalamus (Bregma −0.10).
V. Caruso et al. / Gene 553 (2014) 1–6
V. Caruso et al. / Gene 553 (2014) 1–6
V. Caruso et al. / Gene 553 (2014) 1–6
siRNA was added to the diluted Lipofectamine® RNAiMAX reagent in 1:1 ratio and incubated for 5 min at room temperature. Finally the siRNA–lipid complex was added to the cells in the wells. Untreated wild type cells without any siRNA were used as control. Cells were collected after 48 h of incubation for analysis using Western blot. 2.3.2. Western blot Cells were subjected to lysis by adding 400 μl of lysis buffer (5.4 g urea, 1 ml 10× Hepes buffer pH 8, and one tablet of protease inhibitor cocktail complete Mini EDTA-free (Cat. No. 04693 159 001) (Roche), diluted to 10 ml MilliQ water). Samples were then added to glass beads (equivalent to 50 μl) and subjected to centrifugation in a bullet blender on speed 6 for 1 min + 1 min + 1 min. Samples were separated from glass beads and further centrifuged (Heraeus, Fresco 21; Thermo Scientiﬁc) at 14,000 g at 4 °C for 15 min. Supernatant was collected and protein concentration was determined with a Nano drop. Equal amounts of protein (40 μg) were separated on 12% Mini-Protean TGX gel (Bio-Rad) for GPR162 and reference protein Beta Actin, together with PageRuler prestained protein ladder (Fermentas, Canada) by gel electrophoresis in running buffer (0.1% SDS, 0.025 Tris base, and 0.192 M glycine). The protein bands obtained on the gel were transferred to Immobilon-P PVDF membrane (Millipore) in transfer buffer (0.025 Tris base, 0.192 M glycine, and 20% methanol). Pre-blocking was carried out in blocking buffer (5% nonfat dry milk (Bio-Rad) diluted in 1.5 M NaCl, 0.1 M Tris, 0.05% Tween 20, pH 8.0) for 1 h. Membrane with loaded protein samples was hybridized with the primary antibody against GPR162 (diluted 1: 10, rabbit Anti-GPR162) and Beta Actin (diluted 1:30,000, Mouse anti Beta Actin, Sigma Aldrich, Catalog Number A1978) and incubated overnight at 4 °C. The membrane was further rinsed in water and incubated for 1 h with horseradish peroxidase conjugated secondary antibodies (diluted 1:10,000, goat anti-rabbit and goat anti mouse, Invitrogen). Enhanced chemiluminescent (ECL) method was used for detection. Membrane was incubated for 5 min in a 1:1 mixture of enhancer/luminol and peroxidase buffer solutions (ImmunStar HRP, Bio-Rad). Protein band density was determined by scanning (ChemiDoc Bio-Rad Inc, Hercules, CA, USA) and quantiﬁed using BioRad Laboratories Quantity One 1-D Analysis Software. 2.4. Immunohistochemistry on free ﬂoating sections Floating sections were processed for single immunohistochemistry. Sections were rinsed in TBS and antigen retrieval was performed by heating the sections to 70 °C in 0.01 M citric acid, pH 6.0, (SigmaAldrich, USA) for 20 min followed by a cooling step at room temperature for 30 min. Then, sections were rinsed in TBS, treated for 10 min in 3% H2O2 and 10% methanol (in TBS, pH 7.4) and rinsed in TBS. After 1 h pre-blocking in 2% blocking reagent (Roche Diagnostics, Switzerland) diluted in TTBS (0.1% Tween 20 in Tris-buffered saline), sections were incubated for 24 h at 4 °C in the rabbit GPR162 antibody (1:2000 in Supermix buffer − 0.25% gelatin and 0.5% Triton X-100 in TBS; Innovagen, Sweden). The section was rinsed in TBS and incubated for 1 h in the biotinylated goat anti-rabbit antibody (1:400 in Supermix; Vector, Burlingame, CA) and then in the avidin–biotin complex (1:800 in Supermix; ABC Elite; Vector, Burlingame, CA). Peroxidase was visualized with 0.05% 3,3′-diaminobenzidine tetrahydrochloride (DAB), 0.35% nickel sulfate and 0.01% H2O2 after 10 min incubation. Sections were mounted on gelatin-coated slides, air-dried overnight, dehydrated in ascending concentrations of ethanol, soaked in xylene, mounted in DPX
(Sigma-Aldrich, USA) and analyzed using a Panoramic midi scanner and the Panoramic viewer software v.1.14 (3DHistech, Hungary). 2.5. Fluorescent immunohistochemistry on parafﬁn sections Sections were deparafﬁnized in X-tra Solv (Medite Histotechnik, Germany) and rehydrated through a series of ethanol solutions (100, 95, 75, 50, and 25%) dissolved in H2O, followed by PBS washes. Antigen retrieval was performed by heating the sections to 100 °C in 0.01 M citric acid, pH 6.0, (Sigma) for 10 min. The sections were then washed in PBS, placed in a humidiﬁed chamber, and incubated with primary antibody rabbit anti-GPR162 (custom-made polyclonal antibody, Innovagen, Sweden; dilution 1:100), together with one antibody marker: mouse anti-NeuN (1:400, Millipore, Sweden), chicken anti-GFAP (1:400, Abcam, United Kingdom), mouse anti-Gad67 (1:200, Millipore, Sweden), mouse anti-MAP2 (1:500, SigmaAldrich, USA), mouse anti-pancytokeratin (1:200, Sigma-Aldrich, USA) and chicken anti-NSE (1:100, Abcam, United Kingdom), all diluted in supermix (Tris-buffered saline, 0.25% gelatin, 0.5% Triton X-100) overnight at 4 °C. After washed in PBS, sections were incubated in 1:200 diluted secondary antibodies (donkey anti-rabbit-594, donkey anti-rabbit-488, goat anti-mouse-594, goat antimouse-488, and goat anti-chicken-488 (Invitrogen)) in supermix for 2 h. The sections were further washed in PBS and then stained with DAPI (1:2500) for 5 min prior to mounting with DTG media. Sections were ﬁnally photographed using a ﬂuorescent microscope (Zeiss Axioplan2 imaging, Germany) connected to a camera (AxioCam HRm) with the Carl Zeiss AxioVision version 4.7 Software (Zeiss, Germany). 3. Results and discussion 3.1. Characterization of the GPR162 antibody We generated a custom made rabbit polyclonal antibody and performed Western blot to document the epitope speciﬁcity of our antibody (Fig. 1A). Western blot detected one speciﬁc band between 55 and 72 kDa, consistently with the theoretical size of ~ 64 kDa for the GPR162 protein (NCBI, n.d.). Peptide competition as a negative control was performed and the excess of epitope speciﬁc peptide reduced the binding of the GPR 162 antibody up to the complete removal of the band, showing epitope speciﬁcity of the polyclonal antibody used for our experiment. To further verify the speciﬁcity of the antibody, we performed siRNA knockdown experiments in the neuronal cell-line N25/2, where we knocked down GPR162 expression. Indeed the band at 64 kDa was signiﬁcantly reduced (P b 0.001) with 50% relative to actin expression in the siRNA knockdown cells (Fig. 1B). 3.2. Cell type-speciﬁc expression of GPR162 in the WT mouse brain Single immunohistochemistry with ﬂuorescent markers on WT parafﬁn sections was performed to investigate cell speciﬁcity expression of GPR162 in the mouse brain (Fig. 2). A high degree of co-localization of GPR162 with the neuron-speciﬁc DNA-binding protein marker (NeuN) was found in the hippocampus indicating the expression of GPR162 on both GABAergic as well as other neurons (Mullen et al., 1992). Co-staining with glutamic acid decarboxylase 67 protein (GAD67), a marker for inhibitory GABAergic neurons (Kaufman et al., 1991), revealed the moderate co-expression of GPR162 in both excitatory and non-excitatory neurons in the cortex. Additional colocalization with GPR162 and the neuron-speciﬁc antibody NSE (Crews
Fig. 3. Immunohistochemistry on free ﬂoating sections. Non-ﬂuorescent immunohistochemistry on free ﬂoating brain sections from wild type (WT) mice using custom-made polyclonal GPR162 antibody (Innovagen, Sweden). Overview images (A–E) of sections from WT mice with Bregma coordinates (Franklin and P. G., 2007) in the left corner. Close up images (F–J) with WT sections on the right panel, and with the corresponding annotations on the left panel. (F) GPR162 protein levels are found in cells surrounding the caudate putamen and nucleus accumbens. Higher levels are found in the hippocampus (G), hypothalamus (H), ventral tegmental area (I), and cerebellar lobules (J). Abbreviations and depicted brain regions were adapted from Franklin and Paxinos Brain Atlas 2007 (Franklin and P. G., 2007). (K) Brain sections stained without primary antibody.
V. Caruso et al. / Gene 553 (2014) 1–6
et al., 2008) revealed strong overlapping expression in the hypothalamus, whereas the epithelial cell marker Pan-Cytokeratin (von Overbeck et al., 1985) did not show any overlap in the same area (Bregma level −0.10). No overlap in expression between GPR162 and the astrocytic marker glial ﬁbrillary acidic protein (GFAP) (Reeves et al., 1989) was observed in border between the thalamus and cerebral cortex (Bregma level −2.70). 3.3. GPR162 protein expression in the WT mouse brain The protein expression of GPR162 was investigated through immunohistochemistry in 70 μm coronal mouse brain sections (Fig. 3). Our experiment demonstrated that GPR 162 expression was evident in the hippocampus, particularly in Cornu Ammonis 1-2-3 and dentate gyrus (Fig. 3B) and this might suggest an involvement of the protein in memory and learning (Waltereit and Weller, 2003). However, extensive GPR162 protein expression was found in the hypothalamic nuclei such as arcuate (ARC), lateral (LH) dorsomedial (DMH) and ventromedial (VMH) nucleus known to be involved in the regulation of food intake (Schwartz et al., 2000) (Fig. 3C). A large number of mediators involved in the regulation of food intake are produced in the ARC, which has reciprocal connections with other hypothalamic nuclei, including PVN, VMH, DMH and LH (Schwartz et al., 2000; Valassi et al., 2008). The ARC is located close to median eminence responding to circulating hormones and projecting information to other feeding centers such as the PVN. As reviewed by Stellar, after brain lesion and stimulation studies it has been demonstrated that the ARC projects neurons to LH “the hunger center” and to VMH “the satiety center” (Stellar, 1994) identifying a bi-directional neuronal trafﬁc (Schwartz et al., 2000). Interestingly, similar degree of staining was observed in cells surrounding the nucleus accumbens and amygdala (Fig. 3A and C). These brain regions provide direct and indirect neuronal projections to the hypothalamus (Petrovich, 2013) playing an important role for feeding behavior and reward system (Carter et al., 2013). Further strong staining is found in the ventral tegmental area (Fig. 3D), which is a physiological relevant area implicated in motivation for food and natural reward (Pecina et al., 2003; Mietlicki-Baase et al., 2013). In addition, high GPR162 protein expression was found in the cerebellar Purkinje cell layer, which is known to be constituted by GABAergic neurons (Kayakabe et al., 2013) (Fig. 3E). The novel ﬁnding of our study is that signiﬁcant transcription of GPR162 gene was detected in the hippocampus, hypothalamus, and amygdala, which are brain regions extensively interconnected with each other and involved in the regulation of food intake via reward mechanisms (Ahn and Phillips, 2002; Carlini et al., 2004). Furthermore, GABAergic neurons have been linked to feeding control by multiple studies playing an important role in the regulation of the neuropeptides controlling energy homeostasis (Zeltser et al., 2012). For the ﬁrst time, our results provide direct evidence of high GPR162 expression in the limbic system and expression in GABAergic neurons suggesting a key role in the control of food intake. Acknowledgment This study was supported by the Swedish Research Council, Åhlens Foundation, The Swedish Brain Research Foundation, The Novo Nordisk Foundation, Engkvist foundation and Magnus Bergvall Foundation. References Ahn, S., Phillips, A.G., 2002. Modulation by central and basolateral amygdalar nuclei of dopaminergic correlates of feeding to satiety in the rat nucleus accumbens and medial prefrontal cortex. J. Neurosci. 22, 10958–10965.
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