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Circadian Characteristics of Mice Depleted with GPR7

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Circadian Characteristics of Mice Depleted with GPR7 Biomedical Research 30 (6) 357-364, 2009 Circadian characteristics of mice depleted with GPR7 1, 2 1 1 1 1 3 Naohiro UCHIO , Masao DOI , Masahiro MATSUO , Fumiyoshi YAMAZAKI , Yasutaka MIZORO , Mari HONDO , 3 1, 2 Takeshi SAKURAI and Hitoshi OKAMURA 1 Department of Systems Biology, Graduate School of Pharmacological Science, Kyoto University, Kyoto 606-8501, Japan; 2 Division of Molecular Brain Science, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; 3 Department of Molecular Neurosci- ence and Integrative Physiology, Faculty of Medicine, Kanazawa University, Kanazawa 920-8641, Japan (Received 9 October 2009; and accepted 11 November 2009) ABSTRACT GPR7, now known as a receptor of neuropeptide B and neuropeptide W, is expressed in neurons of the suprachiasmatic nucleus (SCN), the mammalian circadian center. By the quantitative in situ hybridization, we demonstrated that GPR7 mRNA showed a significant circadian rhythm in the SCN showing a peak at early subjective night in both light-dark and constant dark. We character- ized the circadian feature of GPR7-knockout mice, but the period length and the phase-dependent phase shift to light exposure were not disordered in GPR7-knockout mice. Moreover, the food-an- ticipatory behavior in restricted feeding schedule was observed in this gene-deleted mouse similar to wild-type. These results indicate that the role of GPR7 may be subtle or limited in relation to the circadian clock despite its robust expression in the SCN. Most organisms display a physiological and behav- receptor having close structural resemblance to both ioral rhythm with periods of nearly 24 hours. In the somatostatin-3 (SST3) receptor, and the μ-, δ-, mammals, the central circadian oscillator is located and κ-opioid receptors. Recently, its two endoge- in the hypothalamic suprachiasmatic nucleus (SCN) nous ligands, neuropeptide B (NPB) and neuropep- (12). Here the circadian rhythm is generated in the tide W (NPW), were identified (3, 6, 28, 30). oscillator cells by a transcription/translation based Although roles of this receptor on feeding, prolac- autoregulatory negative feedback loop in which the tin-release and pain were reported (29), the roles of protein products of a set of clock genes inhibit their GPR7 on biological rhythms have not been clarified. own transcription (23, 26). In SCN, this cellular In the present study, we demonstrated the circadian rhythm is synchronized through the interaction of change of GPR7 expression in the SCN, and further oscillating cells (32), and entrained to the environ- examined the circadian characteristics of mice delet- mental light-dark cycle by the photic and non-photic ed with GPR7 gene (10). signals (9, 12). Many receptors and their intracellu- lar signal transductions may be enrolled in this in- MATERIALS AND METHODS tercellular communication, and a G-protein-coupled receptor GPR7 might be this candidate, since GPR7 Animals and behavioral rhythm monitoring. For ex- is highly expressed in the SCN (13). GPR7 is origi- amining the expression of GPR7, we purchased nally described by O’Dowd et al. (1995) (21) as a male C57B16 mice at 6 weeks age (JAPS, Osaka, Japan). Mice were housed in 12 h light/dark (LD) cycles (fluorescent light, 300 lux) at least for 2 Address correspondence to: Dr. Hitoshi Okamura Department of Systems Biology, Graduate School of weeks at 22 ± 2°C with freely provided diet and wa- Pharmaceutical Science, Kyoto University, 46-29 Yoshida- ter. Six mice were examined for assessing the brain Shimo-Adachi-cho, Sakyo-ku, Kyoto 606-8501, Japan expression of GPR7 at ZT 16 (ZT stands for Zeitge- Tel: +81-75-753-9552, Fax: +81-75-753-9553 ber time in a LD cycle; ZT0 is light-on and ZT12 is E-mail: [email protected] lights-off). For quantitative analysis of GPR7 in the 358 N. Uchio et al. SCN, mice were examined in the LD and in con- cording to the method detailed previously (27). stant darkness (DD) at every 4 h (n = 4–5 at each Briefly, tissue sections were processed with 1 μg/mL time point for both experiments; total n = 58), start- proteinase K (0.1 M Tris-HCl, pH 8.0, 50 mM ing at ZT0 or CT0 (CT stands for circadian time; EDTA, 10 min) at 37°C and 0.25% acetic anhydride CT0 is subjective dawn and CT12 is subjective in 0.1 M triethanolamine for 10 min. The sections dusk). were then incubated in the hybridization buffer (60% We examined the behavioral characteristics of formamide, 10% dextran sulphate, 10 mM Tris-HCl, GPR7−/− mice in relation to the wild-type mice (10). pH 8.0, 1 mM EDTA, pH 8.0, 0.6 M NaCl, 0.2% Locomotor activity was detected by passive (pyro- N-laurylsarcosine, 500 mg/mL transfer RNA, 1 × electric) infrared sensors (FA-05 F5B; Omron, Kyo- Denhardt’s solution and 0.25% sodium dodecyl to, Japan) (27). Period length was monitored at least sulphate) containing the 33P-UTP-labeled antisense 4 weeks in DD. Moreover, phase-dependent phase- cRNA probes for 16 h at 60°C. Sections for free shift by the brief light exposure, and the effect of floating in situ hybridization were mounted onto restriction feeding (RF) were also examined. Mice gelatin-coated microscope slides, air-dried, and de- were entrained to a 12 h: 12 h LD cycles for at least hydrated. These sections together with 14C-acrylic 1 week before any experimental manipulation. All standards (Amersham, Buckinghamshire, U.K.) were data were monitored and analyzed by Chronobiolo- exposed to Biomax film (Kodak, Rochester, NY) for gy kit (Stanford Software Systems, Stanford, CA). five days and subjected to the image analysis The all experimental procedures were approved by procedure. the Committee for Animal Research of Kobe Uni- versity and the Animal experimentation Committee Quantification of hybridization. The radioactivity of of Kyoto University. each SCN on film was analyzed using a microcom- puter interface to an image analysis system (MCID, In situ hybridization. Mouse GPR7 cDNA fragment Imaging Research, St Catherines, Ontario, Canada) (positions 330–768 (438 bp) of Gene Bank acces- after conversion into the relative optical densities sion no. XM_136404; primers (5’-CGTGTTCAT produced by the 14C-acrylic standards. Data were CCTCAACCTGGCTATC-3’ and 5’-GATGGTGGT normalized with respect to the difference between CACCGGGATGGCAAG-3’) was subcloned into the signal intensities in equal area of the SCN and the pBluescript KS (−) vector (Stratagene, La Jolla, CA) corpus callosum. The intensities of the optical den- and sequenced to verify their identity and orienta- sity of the sections from the rostral to the caudal tion. These cDNA fragment containing vectors were most part of the SCN (8 sections per mouse brain) linearized with restriction enzymes and used as tem- were then summed; the sum was considered to be a plates for antisense cRNA probes. Radiolabeled measure of the amount of GPR7 mRNA in this re- probes for GPR7 mRNA were made using 33P-UTP gion. The intensity values are presented as mean ± (New England Nuclear, Boston, MA) with a stan- SEM (n = 4–5). We indicate relative mRNA abun- dard protocol for cRNA synthesis. dance, which means that the peak intensity value in Mice were deeply anesthetized with ether, and in- the LD or DD condition was adjusted to 100. Data tracardially perfused with 10 mL of ice cold 0.1 M were analyzed by one-way ANOVA followed by phosphate buffer (PB) (pH 7.4), followed by 20 mL Scheffe’s multiple comparisons. of a fixative containing 4% paraformaldehyde in 0.1 M PB. For the mice housed in darkness, anes- Phase-response curve to the light pulse. For the thesia was performed under safe dark red light. The phase response curve, on the last LD cycle, mice re- brains were removed, post-fixed in the same fixative ceived a 30 min light pulse of 300 lux at designated for 24 h at 4°C and placed in 0.1 M PB containing CT times (CT2, CT6, CT10, CT14, CT18, and 20% sucrose for 48 h. These brains were frozen us- CT22). The mice remained in DD for at least 2 ing dry ice and stored at −80°C until use. Mouse week after the pulse. The response to light from brain sections were made 40 μm in thickness by a ZT12–24 or 20–24 were tested by Student’s t-test. cryostat (Leica, Solms, Germany). To minimize The phase response curve was tested by two-way technical variations throughout the hybridization ANOVA. Bonferroni post hoc tests were used for procedure, sections from different experimental con- the comparison between the values of wild-type and ditions were gathered into one group and processed GPR7−/− groups at the same time points. All statisti- simultaneously. In situ hybridization histochemistry cal analyses were performed using GraphPad Prism using the free-floating sections were performed ac- 5.0 (GraphPad Software, La Jolla, CA). GPR7 in mammalian circadian system 359 Entrainment to the food restriction. Age matched sporadic regions of mouse brain (Fig. 1A). In hypo- mice were used. Mice were entrained in 12 h: 12 h thalamus, GPR7 mRNA signals were found at high LD cycle for more than 7 days, and food was given level in the SCN, paraventricular nucleus (PVN), ad libitum before entering RF schedule. Lighting supraoptic nucleus (SON), and hypothalamic dorso- conditions remained the same and water was freely medial nucleus (DMH) (Fig. 1A, a–d). High level available throughout all experiments. On the first signals were also found in central nucleus of the day of RF, food was withdrawn for complete fast- amygdala (AmyCe), insular cortex (InsCx) and zona ing. Then, mice were allowed access to food for 4 h incerta (ZI) in the forebrain (Fig.
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