Dual-Color Single-Cell Imaging of the Suprachiasmatic Nucleus Reveals a Circadian Role in Network Synchrony

•Color-Switch PER2::iLuc is a novel, Cre-inducible mouse reporter for circadian rhythms•Dual-color imaging acquires and unmixes concurrent signals from distinct cell types•SCN AVP and VIP neurons have different functions in network synchrony and stability•Bmal1 has an essential function in AVP neurons for network synchrony in the SCN The suprachiasmatic nucleus (SCN) acts as a master pacemaker driving circadian behavior and physiology. Although the SCN is small, it is composed of many cell types, making it difficult to study the roles of particular cells. Here we develop bioluminescent circadian reporter mice that are Cre dependent, allowing the circadian properties of genetically defined populations of cells to be studied in real time. Using a Color-Switch PER2::LUCIFERASE reporter that switches from red PER2::LUCIFERASE to green PER2::LUCIFERASE upon Cre recombination, we assess circadian rhythms in two of the major classes of peptidergic neurons in the SCN: AVP (arginine vasopressin) and VIP (vasoactive intestinal polypeptide). Surprisingly, we find that circadian function in AVP neurons, not VIP neurons, is essential for autonomous network synchrony of the SCN and stability of circadian rhythmicity. The suprachiasmatic nucleus (SCN) acts as a master pacemaker driving circadian behavior and physiology. Although the SCN is small, it is composed of many cell types, making it difficult to study the roles of particular cells. Here we develop bioluminescent circadian reporter mice that are Cre dependent, allowing the circadian properties of genetically defined populations of cells to be studied in real time. Using a Color-Switch PER2::LUCIFERASE reporter that switches from red PER2::LUCIFERASE to green PER2::LUCIFERASE upon Cre recombination, we assess circadian rhythms in two of the major classes of peptidergic neurons in the SCN: AVP (arginine vasopressin) and VIP (vasoactive intestinal polypeptide). Surprisingly, we find that circadian function in AVP neurons, not VIP neurons, is essential for autonomous network synchrony of the SCN and stability of circadian rhythmicity. In mammals, the hypothalamic suprachiasmatic nucleus (SCN) acts as a master pacemaker controlling circadian rhythms in behavioral states (sleep-wake), feeding behavior, body temperature, and endocrine physiology (Hastings et al., 2018Hastings M.H. Maywood E.S. Brancaccio M. Generation of circadian rhythms in the suprachiasmatic nucleus.Nat. Rev. Neurosci. 2018; 19: 453-469Crossref PubMed Scopus (192) Google Scholar; Saper, 2013Saper C.B. The central circadian timing system.Curr. Opin. 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However, the roles of specific SCN cell types and the intercellular signaling mechanisms engaged in the generation of behavioral circadian rhythms are not fully understood. The study of circadian rhythms requires long timescales (days to weeks) and sensitive detection methods. Pioneered by Millar et al., 1995Millar A.J. Carré I.A. Strayer C.A. Chua N.H. Kay S.A. Circadian clock mutants in Arabidopsis identified by luciferase imaging.Science. 1995; 267: 1161-1163Crossref PubMed Scopus (456) Google Scholar, in vivo bioluminescence imaging has long been used to observe circadian rhythms in real time because of its extremely low background and high signal-to-noise ratio (Troy et al., 2004Troy T. Jekic-McMullen D. Sambucetti L. Rice B. Quantitative comparison of the sensitivity of detection of fluorescent and bioluminescent reporters in animal models.Mol. Imaging. 2004; 3: 9-23Crossref PubMed Scopus (306) Google Scholar; Yamazaki and Takahashi, 2005Yamazaki S. Takahashi J.S. Real-time luminescence reporting of circadian gene expression in mammals.Methods Enzymol. 2005; 393: 288-301Crossref PubMed Scopus (136) Google Scholar). The dynamics of circadian gene expression in the SCN have been studied using real-time imaging of Per1-luciferase and PER2::LUCIFERASE, as well as other transgenic mouse models (Cheng et al., 2009Cheng H.Y. Alvarez-Saavedra M. Dziema H. Choi Y.S. Li A. Obrietan K. Segregation of expression of mPeriod gene homologs in neurons and glia: possible divergent roles of mPeriod1 and mPeriod2 in the brain.Hum. Mol. Genet. 2009; 18: 3110-3124Crossref PubMed Scopus (38) Google Scholar; Kuhlman et al., 2000Kuhlman S.J. Quintero J.E. McMahon D.G. GFP fluorescence reports Period 1 circadian gene regulation in the mammalian biological clock.Neuroreport. 2000; 11: 1479-1482Crossref PubMed Scopus (14) Google Scholar; Mei et al., 2018Mei L. Fan Y. Lv X. Welsh D.K. Zhan C. Zhang E.E. 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Okamura H. Synchronization of cellular clocks in the suprachiasmatic nucleus.Science. 2003; 302: 1408-1412Crossref PubMed Scopus (689) Google Scholar; Yamazaki and Takahashi, 2005Yamazaki S. Takahashi J.S. Real-time luminescence reporting of circadian gene expression in mammals.Methods Enzymol. 2005; 393: 288-301Crossref PubMed Scopus (136) Google Scholar; Yoo et al., 2004Yoo S.H. Yamazaki S. Lowrey P.L. Shimomura K. Ko C.H. Buhr E.D. Siepka S.M. Hong H.K. Oh W.J. Yoo O.J. et al.PERIOD2:LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.Proc. Natl. Acad. Sci. USA. 2004; 101: 5339-5346Crossref PubMed Scopus (1553) Google Scholar). However, circadian genes are ubiquitously expressed, making it difficult to study the circadian properties of specific SCN cell types. This gap in our knowledge motivated us to develop circadian reporter mice that allow conditional genetic manipulation to selectively interrogate subpopulations of SCN neurons. Our novel Color-Switch PER2::LUCIFERASE mouse line uses Cre-lox-dependent reporters that allow the circadian properties of genetically defined cell populations to be studied separately yet simultaneously. Two major classes of neuropeptide-containing neurons, AVP (arginine vasopressin) and VIP (vasoactive intestinal polypeptide), are enriched in the shell and the core regions of the SCN, respectively. There is ample evidence that VIP and VPAC2 receptor (VPAC2R) signaling is important for maintaining the coherence of circadian oscillators both within the SCN and in vivo for the generation of circadian behavioral rhythms (Aton et al., 2005Aton S.J. Colwell C.S. Harmar A.J. Waschek J. Herzog E.D. Vasoactive intestinal polypeptide mediates circadian rhythmicity and synchrony in mammalian clock neurons.Nat. 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Harmar A.J. Okamura H. Hastings M.H. Synchronization and maintenance of timekeeping in suprachiasmatic circadian clock cells by neuropeptidergic signaling.Curr. Biol. 2006; 16: 599-605Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, Maywood et al., 2011Maywood E.S. Chesham J.E. O’Brien J.A. Hastings M.H. A diversity of paracrine signals sustains molecular circadian cycling in suprachiasmatic nucleus circuits.Proc. Natl. Acad. Sci. USA. 2011; 108: 14306-14311Crossref PubMed Scopus (186) Google Scholar). AVP signaling may also mediate interneuronal communication within the SCN, because loss of function of both AVP V1a and AVP V1b receptors profoundly alters the resetting response of mice to shifted light cycles (Yamaguchi et al., 2013Yamaguchi Y. Suzuki T. Mizoro Y. Kori H. Okada K. Chen Y. Fustin J.M. Yamazaki F. Mizuguchi N. Zhang J. et al.Mice genetically deficient in vasopressin V1a and V1b receptors are resistant to jet lag.Science. 2013; 342: 85-90Crossref PubMed Scopus (204) Google Scholar). In addition, deletion of the core clock component, Bmal1, from AVP neurons lengthens the free-running period and enhances re-entrainment to light (Mieda et al., 2015Mieda M. Ono D. Hasegawa E. Okamoto H. Honma K. Honma S. Sakurai T. Cellular clocks in AVP neurons of the SCN are critical for interneuronal coupling regulating circadian behavior rhythm.Neuron. 2015; 85: 1103-1116Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), suggesting that cell-autonomous circadian rhythms in AVP neurons are required to regulate circadian behavioral rhythms (Mieda et al., 2015Mieda M. Ono D. Hasegawa E. Okamoto H. Honma K. Honma S. Sakurai T. Cellular clocks in AVP neurons of the SCN are critical for interneuronal coupling regulating circadian behavior rhythm.Neuron. 2015; 85: 1103-1116Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Deletion of Bmal1 in AVP neurons also reduces the amplitude of circadian rhythms in the dorsal SCN (Mieda et al., 2015Mieda M. Ono D. Hasegawa E. Okamoto H. Honma K. Honma S. Sakurai T. Cellular clocks in AVP neurons of the SCN are critical for interneuronal coupling regulating circadian behavior rhythm.Neuron. 2015; 85: 1103-1116Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar); however, it is unclear how robust these effects are or which cell types are specifically affected. Interestingly, Bmal1 deletion from the liver has no effect on Per2 rhythms, suggesting that external signals (coordinated by the SCN) can compensate for loss of a functioning clock in some tissues (Kornmann et al., 2007Kornmann B. Schaad O. Bujard H. Takahashi J.S. Schibler U. System-driven and oscillator-dependent circadian transcription in mice with a conditionally active liver clock.PLoS Biol. 2007; 5: e34Crossref PubMed Scopus (496) Google Scholar; Lamia et al., 2008Lamia K.A. Storch K.F. Weitz C.J. Physiological significance of a peripheral tissue circadian clock.Proc. Natl. Acad. Sci. USA. 2008; 105: 15172-15177Crossref PubMed Scopus (726) Google Scholar). In the current study, we sought to delineate cell-type-specific functions of the circadian clock in SCN neurons using our novel Color-Switch PER2::LUCIFERASE mouse line. Our results reveal surprising observations about the circadian function of VIP and AVP neurons in SCN network synchrony. To allow for cell-type-specific analysis of circadian rhythms, we generated Color-Switch mice with click beetle red (CBR) luciferase fused to the C terminus of PER2 that switches to click beetle green (CBG) luciferase fusion upon Cre-lox recombination (Gammon et al., 2006Gammon S.T. Leevy W.M. Gross S. Gokel G.W. Piwnica-Worms D. Spectral unmixing of multicolored bioluminescence emitted from heterogeneous biological sources.Anal. Chem. 2006; 78: 1520-1527Crossref PubMed Scopus (59) Google Scholar; Villalobos et al., 2010Villalobos V. Naik S. Bruinsma M. Dothager R.S. Pan M.H. Samrakandi M. Moss B. Elhammali A. Piwnica-Worms D. Dual-color click beetle luciferase heteroprotein fragment complementation assays.Chem. Biol. 2010; 17: 1018-1029Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar; Viviani et al., 2008Viviani V.R. Silva Neto A.J. Arnoldi F.G. Barbosa J.A. Ohmiya Y. The influence of the loop between residues 223–235 in beetle luciferase bioluminescence spectra: a solvent gate for the active site of pH-sensitive luciferases.Photochem. Photobiol. 2008; 84: 138-144PubMed Google Scholar). The targeting construct used to create the Cre-dependent click beetle (CB) luciferase reporter mice is shown in Figure S1. CB luciferase was used for red/green discrimination, because firefly luciferase is pH sensitive and shifts to longer wavelengths so that red/green separation is compromised. The CB luciferases are also about 2×–5× brighter than firefly luciferase (Miloud et al., 2007Miloud T. Henrich C. Hämmerling G.J. Quantitative comparison of click beetle and firefly luciferases for in vivo bioluminescence imaging.J. Biomed. Opt. 2007; 12: 054018Crossref PubMed Scopus (38) Google Scholar). To visualize the red and green bioluminescence signals from PER2::CBR and PER2::CBG, we also developed a dual-color imaging device that is capable of separating and capturing dual-color bioluminescence signals concurrently on the same image-capture device. The bioluminescence signal from the sample was separated with a beam splitter into long wavelengths (>565 nm) and short wavelengths (<565 nm). The long- and short-wavelength signals were filtered with a long-pass filter (>625 nm) and a short-pass filter (<550 nm), respectively, to separate the green and red bioluminescence signals. The green- and red-channel images were then focused onto the right and left halves of a cooled charge-coupled device (CCD) camera detector so that the red and green channels could be collected simultaneously frame by frame (Figure 1A). We next crossed Color-Switch PER2::LUCIFERASE mice with VIP-ires-Cre (Taniguchi et al., 2011Taniguchi H. He M. Wu P. Kim S. Paik R. Sugino K. Kvitsiani D. Fu Y. Lu J. Lin Y. et al.A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex.Neuron. 2011; 71: 995-1013Abstract Full Text Full Text PDF PubMed Scopus (969) Google Scholar) or AVP-ires-Cre (Pei et al., 2014Pei H. Sutton A.K. Burnett K.H. Fuller P.M. Olson D.P. AVP neurons in the paraventricular nucleus of the hypothalamus regulate feeding.Mol. Metab. 2014; 3: 209-215Crossref PubMed Scopus (71) Google Scholar) mouse lines, and ex vivo cultures of SCN explants were prepared (Yamazaki and Takahashi, 2005Yamazaki S. Takahashi J.S. Real-time luminescence reporting of circadian gene expression in mammals.Methods Enzymol. 2005; 393: 288-301Crossref PubMed Scopus (136) Google Scholar). As expected, VIP cells with PER2::CBG expression were localized in the ventral/core region of the SCN close to the optic chiasm (Figure 1B; Video S1), whereas AVP cells were localized in the dorsal/shell region of the SCN (Figure 1C; Video S2). Heatmap representations of single-cell rhythms from VIP (Figure 1D) and AVP (Figure 1E) neurons show that circadian rhythmicity was coherent in SCN explants, similar to that seen previously in pan-SCN PER2::LUC recordings (Buhr et al., 2010Buhr E.D. Yoo S.H. Takahashi J.S. Temperature as a universal resetting cue for mammalian circadian oscillators.Science. 2010; 330: 379-385Crossref PubMed Scopus (537) Google Scholar; Ko et al., 2010Ko C.H. Yamada Y.R. Welsh D.K. Buhr E.D. Liu A.C. Zhang E.E. Ralph M.R. Kay S.A. Forger D.B. Takahashi J.S. Emergence of noise-induced oscillations in the central circadian pacemaker.PLoS Biol. 2010; 8: e1000513Crossref PubMed Scopus (140) Google Scholar; Liu et al., 2007Liu A.C. Welsh D.K. Ko C.H. Tran H.G. Zhang E.E. Priest A.A. Buhr E.D. Singer O. Meeker K. Verma I.M. et al.Intercellular coupling confers robustness against mutations in the SCN circadian clock network.Cell. 2007; 129: 605-616Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). Importantly, the SCN slice is not in steady state when the tissue slice is first prepared. Initially, the SCN slice reflects a state of the oscillator system that is similar to what it was previously in vivo. As documented in many studies, the relative phase coherence of SCN oscillators is regulated by photoperiod, and this can be seen subsequently in SCN slices as the coherence of phases of the population of cells in the SCN (Buijink et al., 2016Buijink M.R. Almog A. Wit C.B. Roethler O. Olde Engberink A.H. Meijer J.H. Garlaschelli D. Rohling J.H. Michel S. Evidence for Weakened Intercellular Coupling in the Mammalian Circadian Clock under Long Photoperiod.PLoS ONE. 2016; 11: e0168954Crossref PubMed Scopus (27) Google Scholar; Schaap et al., 2003Schaap J. Albus H. VanderLeest H.T. Eilers P.H. Détári L. Meijer J.H. Heterogeneity of rhythmic suprachiasmatic nucleus neurons: Implications for circadian waveform and photoperiodic encoding.Proc. Natl. Acad. Sci. USA. 2003; 100: 15994-15999Crossref PubMed Scopus (166) Google Scholar; VanderLeest et al., 2007VanderLeest H.T. Houben T. Michel S. Deboer T. Albus H. Vansteensel M.J. Block G.D. Meijer J.H. Seasonal encoding by the circadian pacemaker of the SCN.Curr. Biol. 2007; 17: 468-473Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). With time in vitro, the SCN slices relax to a new steady state with respect to mutual coupling of the population of oscillators. During this first week in culture, the phase relationships among SCN neurons gradually change, and SCN neurons can exhibit different period lengths as the slice goes from one state (from in vivo) to a new steady state (to in vitro). With our methods, we can directly measure these subtle period changes during this transition interval. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJkMjBlN2Q4NTJmNmM3OTdlODI5ODgyMzBhYzJlMDQ2YSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjM1NDc0NTM1fQ.S5X4IkfEcLI7RXAcF_O53IZ-Y_l4ZtpFcyGAWlGX_6pEsGgfwQkvjMSQ6XYbKYrdkxP1CeL9UYb92DIiNhDSAOsrjRiTRH0DEQks2Sl3PabSHde-A80ofJ2APe-_2Ea1wiAOL8auT56-Ew65SE6eKS8q0YCiL8wbSmJXmSkuWtRuqE97f-r4Xmf74WOhnizl7y23piO1YvBo4NkSH3aOA1pgHNe6Z5KpQVNt9GhpOgUF4ryoj4rWV6FWKBjwLSfFxAktfQQQ4D-evmtNPThkDJtG_4KWrb2mh3ePIitwPlVYfASi9BXHzf2OedKQv_lKu8uLswxfWJOcTTZEQlJmpg Download .mp4 (1.29 MB) Help with .mp4 files Video S1. Dual-Color, Time-Lapse Imaging of a VIP-Cre SCN, Related to Figure 1 eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI3MmE4ZDBkODA5YWY0YWEzNTZmNzU4NzVlNmFkMjc1NCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjM1NDc0NTM1fQ.VVpQrn7L1BFJJ6fsKgJn8ArnmEKPKr3qjGCG94dnFZYYetqpEMD0s995CcrCQYYt5TkhUE4f1wRqeS4TgGd4QeJar4zZRg6B98RujPzOMwPH3WiuXo7-hMOig8KxujCt22JJGM14R_R6Qd77GAzLoDVAU2HOSgS6CivzjkzknEWtizsO0BMTbWE0PUX5Qw81LnklWtXDk7EGC5u3OqHS7FeoB5E1Dx7r0SnEQNoub9A7xtXfce4QzgkOQIsgMaFv_MPy5eFl4k7WWWL80frZ-HFm4BVQSB_0rYzgtZjuUpw8Bp7VAdbMCKsPuE43sohi2M3ygA4igSdzFw_x-L59Mw Download .mp4 (0.98 MB) Help with .mp4 files Video S2. Dual-Color, Time-Lapse Imaging of an AVP-Cre SCN, Related to Figure 1 To evaluate the periodicity of genetically identified single cells from dual-color imaging, bioluminescence time series were analyzed with a custom-made pipeline (Figure S2; see STAR Methods). VIP neurons had a delayed average phase and longer average single-cell period compared with non-VIP cells (Figure 1F; Figure S3). In contrast, AVP neurons had a shorter average single-cell period length but no average phase difference compared with non-AVP cells (Figure 1G; Figure S3). Because both VIP and AVP cell types used PER2::CBG rather than PER2::CBR, the red/green reporters cannot account for the average period differences seen between VIP and AVP SCN neurons. These results suggest that our new mouse line allows accurate measurement of circadian PER2::LUC rhythms from any genetically accessible cell type given an appropriate Cre driver line. As with the original PER2::LUC reporter mouse (Yoo et al., 2004Yoo S.H. Yamazaki S. Lowrey P.L. Shimomura K. Ko C.H. Buhr E.D. Siepka S.M. Hong H.K. Oh W.J. Yoo O.J. et al.PERIOD2:LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.Proc. Natl. Acad. Sci. 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Tran H.G. Zhang E.E. Priest A.A. Buhr E.D. Singer O. Meeker K. Verma I.M. et al.Intercellular coupling confers robustness against mutations in the SCN circadian clock network.Cell. 2007; 129: 605-616Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar; Tokuda et al., 2015Tokuda I.T. Ono D. Ananthasubramaniam B. Honma S. Honma K. Herzel H. Coupling Controls the Synchrony of Clock Cells in Development and Knockouts.Biophys. J. 2015; 109: 2159-2170Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). To study the neuropeptide-specific roles of neurons within this network, we applied tetrodotoxin (TTX), a sodium ion channel blocker, to inhibit the firing of action potentials and thus disrupt the coupling of SCN neurons (Yamaguchi et al., 2003Yamaguchi S. Isejima H. Matsuo T. Okura R. Yagita K. Kobayashi M. Okamura H. Synchronization of cellular clocks in the suprachiasmatic nucleus.Science. 2003; 302: 1408-1412Crossref PubMed Scopus (689) Google Scholar). Neuronal firing results in the release of AVP and VIP in the SCN (Mazuski et al., 2018Mazuski C. Abel J.H. Chen S.P. Hermanstyne T.O. Jones J.R. Simon T. Doyle 3rd, F.J. Herzog E.D. Entrainment of Circadian Rhythms Depends on Firing Rates and Neuropeptide Release of VIP SCN Neurons.Neuron. 2018; 99: 555-563Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar; Pennartz et al., 1998Pennartz C.M. Bos N.P. Jeu M.T. Geurtsen A.M. Mirmiran M. Sluiter A.A. Buijs R.M. Membrane properties and morphology of vasopressin neurons in slices of rat suprachiasmatic nucleus.J. Neurophysiol. 1998; 80: 2710-2717Crossref PubMed Scopus (27) Google Scholar); therefore, blocking neuronal firing via TTX prevents intercellular communication within the SCN. Washout of TTX reverses the decoupling of the network, resulting in restored synchrony and high-amplitude circadian rhythmicity (Abel et al., 2016bAbel J.H. Meeker K. Granados-Fuentes D. St John P.C. Wang T.J. Bales B.B. Doyle 3rd, F.J. Herzog E.D. Petzold L.R. Functional network inference of the suprachiasmatic nucleus.Proc. Natl. Acad. Sci. USA. 2016; 113: 4512-4517Crossref PubMed Scopus (31) Google Scholar; Yamaguchi et al., 2003Yamaguchi S. Isejima H. Matsuo T. Okura R. Yagita K. Kobayashi M. Okamura H. Synchronization of cellular clocks in the suprachiasmatic nucleus.Science. 2003; 302: 1408-1412Crossref PubMed Scopus (689) Google Scholar). To examine the respective roles of VIP and AVP neurons in maintaining synchrony within the SCN network, we also compared the effects of TTX on VIP or AVP neurons in a Cre-only or Bmal1 conditional deletion genetic background. The VIP-Cre and AVP-Cre mouse lines were crossed to Color-Switch mice and a Cre-lox conditional Bmal1 knockout mouse line (Johnson et al., 2014Johnson B.P. Walisser J.A. Liu Y. Shen A.L. McDearmon E.L. Moran S.M. McIntosh B.E. Vollrath A.L. Schook A.C. Takahashi J.S. Bradfield C.A. Hepatocyte circadian clock controls acetaminophen bioactivation through NADPH-cytochrome P450 oxidoreductase.Proc. Natl. Acad. Sci. USA. 2014; 111: 18757-18762Crossref PubMed Scopus (48) Google Scholar) to produce VIP-Cre; Bmal1fx/fx; Per2iLuc/iLuc (called VIP-Bmal1−/−) (Table S1), and AVP-Cre; Bmal1fx/fx; Per2iLuc/iLuc (called AVP-Bmal1−/−) (Table S1) mice. VIP-Cre and AVP-Cre lines were used as heterozygous drivers to mitigate potential hypomorphic expression of their peptide gene products. In these mice, cells with green bioluminescence signals also indicate the excision of Bmal1 and loss of cell-autonomous circadian function. We first compared TTX-mediated synchronization between VIP-Cre-labeled SCN and VIP-Bmal1−/− SCN, because VIP is known to play an important role in SCN synchrony (Aton et al., 2005Aton S.J. Colwell C.S. Harmar A.J. Waschek J. Herzog E.D. Vasoactive intestinal polypeptide mediates circadian rhythmicity and synchrony in mammalian clock neurons.Nat. Neurosci. 2005; 8: 476-483Crossref PubMed Scopus (552) Google Scholar). In both VIP-Cre and VIP-Bmal1−/−, individual cells exhibited high-amplitude oscillations with moderate damping. When 1 μM TTX was added, the amplitude of single cells and the overall SCN damped quickly and the phase coherence was disrupted (Figures 2A–2F; Figure S3), as seen previously (Abel et al., 2016bAbel J.H. Meeker K. Granados-Fuentes D. St John P.C. Wang T.J. Bales B.B. Doyle 3rd, F.J. Herzog E.D. Petzold L.R. Function