Synchronous circadian voltage rhythms with asynchronous calcium rhythms in the suprachiasmatic nucleus

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1 Syncronous circadian voltage rytms wit asyncronous calcium rytms in te supraciasmatic nucleus Ryosuke Enoki a,b,c,1,2, Yosiaki Oda a,b,1, Miciiro Mieda d, Daisuke Ono a,3, Sato Honma b,4, and Kenici Honma b a Potonic ioimaging Section, Researc enter for ooperative Projects, Hokkaido University Graduate Scool of Medicine, Sapporo , Japan; b Department of ronomedicine, Hokkaido University Graduate Scool of Medicine, Sapporo , Japan; c Precursory Researc for Embryonic Science and Tecnology, Japan Science and Tecnology Agency, Saitama , Japan; and d Department of Molecular Neuroscience and Integrative Pysiology, Graduate Scool of Medical Sciences, Kanazawa University, Kanazawa, Isikawa , Japan Edited by Josep S. Takaasi, Howard Huges Medical Institute, University of Texas Soutwestern Medical enter, Dallas, TX, and approved February 17, 2017 (received for review October 10, 2016) Te supraciasmatic nucleus (SN), te master circadian clock, contains a network composed of multiple types of neurons wic are tougt to form a ierarcical and multioscillator system. Te molecular clock macinery in SN neurons drives membrane excitability and sends time cue signals to various brain regions and periperal organs. However, ow and at wat time of te day tese neurons transmit output signals remain largely unknown. Here, we successfully visualized circadian voltage rytms optically for many days using a genetically encoded voltage sensor,. Unexpectedly, te voltage rytms are syncronized across te entire SN network of cultured slices, wereas simultaneously recorded rytms are topologically specific to te dorsal and ventral regions. We furter found tat te temporal order of tese two rytms is celltype specific: Te rytms paselead te voltage rytms in AVP neurons but and voltage rytms are nearly in pase in VIP neurons. We confirmed tat circadian firing rytms are also syncronous and are coupled wit te voltage rytms. Tese results indicate tat SN networks wit asyncronous rytms produce coerent voltage rytms. circadian rytm membrane potential intracellular calcium timelapse imaging neuronal network In mammals, daily rytms in biocemistry, pysiology, and beavior are coordinated by te master circadian clock located in te ypotalamic supraciasmatic nucleus (SN) in te brain (1). In rats and mice te SN consists of 20,000 neurons, eac of wic demonstrates selfsustained circadian oscillations (2, 3). In individual SN neurons, cellular circadian rytms are generated by an autoregulatory transcriptional and translational feedback loop (core loop) consisting of te clock genes Period (Per) 1, Per2, ryptocrome (ry) 1, ry2, mal1, and lock and teir protein products (4). Tese cellular clocks regulate membrane excitability and define firing patterns in te SN neurons (5). In vivo and in vitro electropysiological studies revealed tat rytms in resting membrane potentials and te frequency of action potential firings are ig during te subjective day and low during te subjective nigt (5). lockade of neuronal firings by tetrodotoxin () leads to te desyncronization of te SN network in vitro (6, 7) and beavioral arrytmicity in vivo (8). Tese studies indicate tat output signals of neuronal firings in te SN are crucial not only for intercellular communications witin te SN network but also for conveying time cues to syncronize rytms in te periperal tissues. At te network level, bioluminescence and fluorescence imaging revealed te topologically specific patterns of rytms in te expression of clock genes suc as Per1 (6) and PER2 (9) and te intracellular concentration (7) in te dorsal to ventral SN regions. It as been proposed tat tere is more tan one regional oscillator in te SN and tat witin tese regional oscillators a group of oscillating neurons syncronize wit eac oter and beave differently from groups in oter regional oscillators (10, 11). Tese groups include te oscillators in te dorsal SN were argininevasopressin (AVP) neurons are abundant and in te ventral SN were vasoactive intestinal peptide (VIP) neurons predominate (10, 12 15). To examine ow and were different SN neurons regulate membrane excitability and send output signals, we need to record neuronal activities on a large scale at a singlecell resolution for several days. onventional electropysiology tecniques, suc as patcclamp recording, allow us to monitor only a few neurons for few ours. Extracellular recordings, suc as multiunit activity (16 19) and multielectrode array dis (MED) recordings (20), ave limited spatial resolution. Recent advances in imaging metods enable us to record neuronal firing patterns optically in vitro and in vivo (21). However, a subtresold membrane excitation or depolarizing state tat does not cange te firing rate is believed to contribute to information processing in te brain (22, 23). Indeed, daily silencing of neuronal firing by SN neurons as been reported (24). Tus, tere is a need to measure membrane potentials in master circadian clock neurons directly. Significance Te mammalian master circadian clock, te supraciasmatic nucleus (SN), contains a network composed of various neuron types. Te SN network plays critical roles in expressing robust circadian rytms in pysiology and beavior, suc as sleep wake cycles. Te molecular clock in individual SN neurons controls membrane excitability, and sends output signals to various organs. However, ow te SN neurons transmit output signals remains unknown. Using a genetically encoded voltage sensor, we directly measured te circadian rytms of membrane voltage in te SN network. Remarkably, te circadian voltage rytms are syncronous across te entire SN network, wereas simultaneously recorded rytms are asyncronous in te dorsal and ventral SN regions. Tese results indicate tat te SN network produces coerent output signals. Autor contributions: R.E., Y.O., S.H., and K.H. designed researc; R.E., Y.O., and D.O. performed researc; M.M. contributed new reagents/analytic tools; R.E. and Y.O. analyzed data; and R.E., S.H., and K.H. wrote te paper. Te autors declare no conflict of interest. Tis article is a PNAS Direct Submission. 1 R.E. and Y.O. contributed equally to tis work. 2 To wom correspondence sould be addressed. enoki@pop.med.okudai.ac.jp. 3 Present address: Department of Neuroscience II, Researc Institute of Environmental Medicine, Nagoya University, Nagoya , Japan. 4 Present address: Researc and Education enter for rain Science, Hokkaido University, Sapporo , Japan. Tis article contains supporting information online at /pnas //DSupplemental. E2476 E2485 PNAS Publised online Marc 7,

2 Over te last 40 y, since an organic voltage probe was first applied in te central nervous system (25), direct optical measurement of membrane potentials as been a goal in neuroscience. However, te voltage probe ad drawbacks; its signal was very small compared wit tat of oter probes, suc as probes. Neverteless, te recent improvement in fluorescent proteinbased voltage sensors enabled us to record neuronal activities accurately in real time (26, 27). Voltage sensors ave te important advantage of being able to be specifically expressed in an individual cell type in te brain for several days. We recently developed igresolution timelapse imaging metods to visualize te spatiotemporal dynamics of intracellular in te SN network for many days witout noticeable potoinduced toxicity or bleacing. Tus, all te tools necessary for direct monitoring of membrane potentials are now available. In tis study, we successfully measured te circadian rytms of membrane voltage in te neurons of cultured SN using a genetically encoded voltage sensor, (27), and igresolution confocal imaging microscopy (7, 28). Using (29), a red indicator, and celltype specifically expressed, we successfully demonstrated te rytmic cange in membrane voltage and rytms simultaneously in te SN network. Unexpectedly, circadian voltage rytms are syncronous trougout te entire SN network, wereas rytms are topologically specific to te dorsal and ventral regions in te same SN slice. Tese findings provide an important insigt into ow te SN network encodes circadian time in vivo. Results Expression Patterns of a Genetically Encoded Voltage Sensor,. To elucidate te spatiotemporal patterns of membrane potentials in SN neurons, was transfected in cultured SN slices of newborn mice [postnatal day (P) 4 6] using a recombinant adenoassociated virus (AAV) under a neuronspecific promoter, uman synapsin I (SynI) (Fig. S1). Fig. 1A sows representative expression patterns of fluorescence in te SN prepared from 57L/6J mice. Te expression pattern of in te SN network was distinct from tat of te probes, suc as Yellow ameleon 3.60 (7, 28) and GaMP6s (30). signals were detected in te plasma membrane of soma and fibers (i.e., axon and dendrites) but not in te cytosol or nucleus (Fig. 1A, Lower). ecause is expressed trougout te plasma membrane, including te axon and dendrites, it is difficult to identify te origin of te signals. To overcome tis difficulty, te SN slices from VIP and AVPre mice were transfected wit AAVencoding SynIFlex. elltype specific expression of allowed us to identify te VIP (Fig. 1) andavp(fig.1) neurons separately and to estimate te distribution of fluorescence in dendrites and axons. Te VIP neurons wose cell bodies were localized in te ventral SN sent fibers to te dorsal SN region (Fig. 1), wereas te AVP neurons wit cell bodies located mostly in te dorsal SN sent fibers to te entire SN (Fig. 1). elltype specific expressions of in te VIP and AVP neurons were confirmed by immunoistocemical staining against VIP and AVP neuropeptides, respectively (Fig. S2). Simultaneous Recording of ircadian Rytms in Voltage and Intracellular alcium. Using togeter wit, we successfully detected te circadian rytms in membrane potential (voltage rytm) and intracellular levels ( rytm) simultaneously in te SN (Fig. 2). Remarkably, we found tat te voltage rytms were syncronous trougout te entire SN network. To quantify te rytms at te level of te SN network, we created acropase (peak pase) maps using a pixelbased analysis program as described previously (7, 30, 31). Te acropase maps, expressed relative to te slice mean, sowed tat te voltage rytms were nearly in pase trougout te entire SN region (sown in yellowgreen colors) (Fig. 2, Left). On te oter and, te rytms were topologically specific, wit an advanced pase in te dorsal region (sown in cold colors) and a delayed pase in te center and ventral regions (sown in warm colors) (Fig. 2, Rigt), as previously reported (7). We statistically compared te regional difference between representative areas (eac μm) in te dorsal and ventral SN. Te mean acropase of te voltage rytm was not significantly different in tese areas, indicating tat tey were syncronous (12.4 ± 0.5 and 12.0 ± 0.5, respectively; n = 6; P = 0.61) (Fig. 2, Left), wereas te mean acropase of te rytm in tese areas was significantly different, indicating topological specificity (9.1 ± 0.3 and 13.6 ± 0.4, respectively; n = 6; P < 0.001) (Fig. 2, Rigt). We compared te pase difference between te voltage and rytms in tese regions. Te rytm was paseadvanced relative to te voltage rytms in te dorsal region (2.6 ± 0.4 ) (P < 0.001), but tese two rytms were nearly in pase in te ventral region ( 0.3 ± 0.3 ) (P = 0.62) (Fig. 2 D and E). Signal Origin of. To validate te signal origin and dynamic range of fluorescence in our experimental conditions, we manipulated te membrane potentials of SN neurons by canging extracellular concentrations of potassium ion (0.5, 0.3, 5.4, and 10 mm Kl), as reported previously (32). We recorded te resting membrane potentials and action potentials by NEUROSIENE PNAS PLUS A SynI, 57L/6J SynIFlex, VIPre SynIFlex, AVPre Dorsal Ventral Dorsal Ventral Dorsal Ventral 10 m 10 m 10 m Fig. 1. Expression patterns of te voltage probe in te SN network. Expression patterns of fluorescence in te entire SN (Upper) and te dorsal/ventral regions (ROIs marked by red squares in te upper panel) (Lower) of te cultured SN slices from 57L/6J mice (A), VIPre mice (), and AVPre mice ()., tird ventricle;, optic ciasm. Enoki et al. PNAS Publised online Marc 7, 2017 E2477

3 A D E Fig. 2. Simultaneous recordings of te voltage and rytms. (A) Expression patterns of (Left) and (Rigt) fluorescence at te peak circadian pase. Te estimated border of te SN is indicated by a dased line. () Acropase maps of te voltage rytms (Left) and rytms (Rigt). Mean acropase of te entire SN regions was separately normalized to zero for voltage and rytms. olor bars indicate te relative time of day (ours). () Te mean acropase of te circadian voltage (Left) and (Rigt) rytms from two regions in te SN (square ROIs in A). For a regional comparison of te rytms, μm ROIs in te top tird (near te tird ventricle) and bottom twotirds (near te optic ciasma) were selected as te dorsal and ventral regions, respectively. A significant pase difference was detected between te dorsal (d) and ventral (v) regions in te rytms but not in te voltage rytms. (D) Raw (Upper) and 24 detrended (Lower) traces of te voltage rytms (green traces) and rytms (red traces) in te dorsal (1) and ventral (2) SN from te square ROIs in A. Te circadian peak is indicated by arroweads of eac color. Detrended data were smooted wit a 3 moving average metod. (E) Te pase difference (ΔPase) between te voltage and rytms in te dorsal and ventral SN (six slices). All data are given as te mean ± SEM; ***P < a.u., arbitrary units. Note tat in all figures te vertical scale bar was inverted because fluorescence indicates tat te fluorescence dims upon depolarization of te plasma membrane, reported to be a caracteristic feature of (27). wolecell patcclamp recordings from single SN neurons (23 neurons in six slices) and detected te fluorescence canges in in six SN slices. We confirmed tat reports canges in te membrane potentials wit a dynamic range from 50 mv to 30 mv under tis condition (Fig. S3). Te mean membrane potentials were almost identical wit or witout firings (Fig. S3). Te estimated range of circadian oscillation was 6.26 ± 1.43 mv in te dorsal region and 6.35 ± 1.36 mv in te ventral region, a difference tat was not statistically significant (P = 0.96) (Fig. S3 D and E). We concluded tat signals reflect te cange in te resting membrane potential, not te firing level. elltype Specific Recording of te Voltage Rytms. Voltage rytms in VIP neurons. Fig. 3 sows celltype specific monitoring of te voltage rytms in te VIP neurons. Te voltage rytms were detectable mostly in te ventral region and were absent in te dorsal region near te tird ventricle (Fig. 3, Left). Tey were in pase trougout te SN. Te rytms, monitored in entire SN neurons by, were localized across te dorsal to te ventral SN (Fig. 3, Rigt), as reported previously (7, 30). Te voltage and rytms in te ventral SN were nearly in pase ( 0.6 ± 0.3, n = 9 slices). To examine te roles of te SN neural network in te circadian and rytms, we tested te effects of blockers for voltagedependent fast Na + and cannels. Te amplitude of te circadian rytms in terms of a peak troug difference was compared before (pretreatment) and after te drug application. To confirm te circadian rytmicity, te fluorescence signals were fitted to a cosine curve using a leastsquare regression metod, and te goodness of fit was statistically evaluated (P < by percent rytm). Eac blocker differentially affected te amplitude of te two rytms. A fast Na + cannel blocker, (1 μm), reduced te amplitudes of te voltage rytms (30.8 ± 13.7% of te pretreatment level; P = 0.037) and of rytms (32.1 ± 12.4% of te pretreatment level; P = 0.032) in te VIP neurons (n = 3 slices). However, te voltage and levels still exibited statistically significant circadian rytms even after treatment (Fig. 3 and F). Tese results indicate te presence of circadian voltage and rytms independent of neuronal firings in VIP neurons. E Enoki et al.

4 A F G D E NEUROSIENE PNAS PLUS Fig. 3. Spatiotemporal profiles of te voltage and rytms in VIP neurons. (A) Expression patterns of in te VIP neurons (Left) and in te entire SN (Rigt). () Acropase maps of te voltage rytms (Left) and te rytms (Rigt). Mean acropase of te entire SN regions was separately normalized to zero for voltage and rytms. ( E) Representative circadian voltage rytms (green traces) and rytms (red traces). Te upper and lower traces in eac panel sow raw and 24 detrended data (smooted wit a 3 moving average), respectively. () FastNa + cannel blocker (1 μm ) (n = 3). (D) Ltype cannel blocker (3 μm nimodipine) (n = 3). (E) oapplication of te fast Na + cannel blocker (1 μm ) and te Ltype cannel blocker (3 μm nimodipine) (n = 3). (F) Mean amplitudes of te and voltage rytms after application of te respective cannel blockers expressed as te percent of te pretreatment level. A onesample t test was used to validate te blocker effects. (G) Pase differences (ΔPase) between te voltage and rytms after application of te respective cannel blockers (n = 3 in eac condition). A paired t test was used to validate te blocker effects. *P < 0.05; **P < 0.01; ***P < All data are given as te mean ± SEM. cannels are known to contribute to te membrane potential (32 35). Particularly, Ltype cannels are reported to be te primal source in te SN (34, 36). Indeed, we found tat an Ltype cannel blocker, nimodipine (3 μm), significantly suppressed bot te voltage rytms (40.9 ± 10.2% of te pretreatment level; P = 0.028) and rytms (41.2 ± 2.2% of te pretreatment level; P = ) (n = 3 slices) (Fig. 3 D and F). A blocker mixture of and nimodipine greatly suppressed te amplitude of te rytms (6.1 ± 0.2% of te pretreatment level; P < 0.001), but te effect was less profound in te voltage rytms (45.0 ± 7.1% of te pretreatment level; P = 0.016) (n = 3 slices) (Fig. 3 E and F). Tese results indicate tat fast Na + cannels and Ltype cannels contribute additively to te rytms, but te two cannels contribute redundantly to te voltage rytms. Tese findings suggest tat te voltage rytms are independent of te rytms in te VIP neurons. We ten analyzed te effects of blockers on te pase relationsip in te VIP neurons. Pase differences between te and voltage rytms were uncanged after te application of (0.8 ± 0.5, P = 0.24) (n = 3 slices) or nimodipine (0.6 ± 1.1, P = 0.61) (n = 3 slices). However, tey became more variable after coapplication of and nimodipine (1.0 ± 4.6, P = 0.85) (n = 3 slices) (Fig. 3G). Voltage rytms in AVP neurons. We conducted te same series of experiments in te AVP neurons (Fig. 4). As sown in Fig. 1, te cell bodies of te AVP neurons located in te dorsal SN sent fibers to te entire SN, including te ventral region. We found tat te voltage rytms of te AVP neurons were syncronous in large areas of te SN. expressed in te entire SN revealed tat te rytms were topologically specific in te dorsal to ventral SN (Fig. 4, Rigt). Te rytms were paseadvanced relative to te voltage rytms, and te pase difference between te two rytms was 3.1 ± 0.5 in te AVP neurons (n = 9 slices). Application of 1 μm significantly reduced te amplitude of te rytms (58.5 ± 10.9% of te pretreatment amplitude, P = 0.032) but did not cange te amplitude of te voltage rytms (119.8 ± 20.0%; P = 0.39) (n = 3 slices) (Fig. 4 and G). Similarly, nimodipine (3 μm) reduced te amplitude of te rytms (41.2 ± 2.2%; P < 0.001) but did not cange te amplitude of te voltage rytms (159.9 ± 42.8%; P = 0.26) (n = 3slices)(Fig.4D). oapplication of and nimodipine reduced te amplitude of bot rytms (17.5 ± 4.8%, P = ) and voltage rytms Enoki et al. PNAS Publised online Marc 7, 2017 E2479

5 A in AVP neurons Peak in entire SN Peak raw signals 210 a.u Acropase Map in AVP neurons in entire SN 260 a.u D Vm detrended time () raw signals Nimodipine F Amplitude (% of pretreat) * Nimo &Nimo * G * *** ** Nimo &Nimo Difference in Pase () (pretreatduring treatment) Nimo &Nimo E Vm detrended time () raw signals & Nimodipine Vm detrended time () Fig. 4. Spatiotemporal profiles of te voltage and rytms in AVP neurons. (A) Expression patterns of in te AVP neurons (Left) and in te entire SN (Rigt). () Acropase maps of te voltage rytms (Left) andte rytms (Rigt). Mean acropase of te entire SN regions was separately normalized to zero for voltage and rytms. ( E) Representative circadian voltage rytms (green traces) and rytms (red traces). ( and D) Application of 1 μm (n = 3) () or3μm nimodipine(n = 3) (D). (E) oapplication of (1 μm) and nimodipine (3 μm) (n = 3). (F) Mean amplitudes of te and voltage rytms after application of te respective cannel blockers expressed as te percentage of te pretreatment level. A onesample t test was used to validate te blocker effects. (G) Differences in pase (ΔPase) between te voltage and rytms after te application of te respective cannel blockers (n = 3 in eac condition). A paired t test was used to validate te blocker effects. *P < 0.05; **P < 0.01; ***P < All data are given as te mean ± SEM. (45.5 ± 9.0%; P = 0.026) (n = 3 slices) (Fig. 4D). Tese results indicate tat te rytms were mediated by te activation of fast Na + and Ltype cannels in te AVP neurons, wereas te voltage rytms were mediated by a and nimodipineinsensitive mecanism. Te suppression of te voltage rytms by coapplication of and nimodipine could be caused by te reduced inputs from te damped VIP rytms (Fig. 3F). Te pase differences between te rytms and voltage rytms were uncanged on te first cycle (4.2 ± 1.7 ; P = 0.13) but were canged significantly on te second cycle (5.8 ± 1.1 ; P = 0.03) after application (n = 3 slices). On te oter and, te application of nimodipine (2.6 ± 2.4 ; P = 0.39) (n = 3 slices) or te coapplication and nimodipine (3.2 ± 4.6 ; P = 0.55) (n = 3 slices) did not cange te pase difference (Fig. 4G). We confirmed tese findings by double transfection of AAVs encoding SynIFlex and SynIFlexj (37) in te SN from VIPre and AVPre mice. Te circadian rytms were in pase wit te voltage rytms in te VIP neurons ( 0.5 ± 0.3 ; n = 3 slices) (Fig. S4 A and ) but were paseadvanced by 6.6 ± 1.4 (n = 3 slices) relative to te voltage rytms in te AVP neurons (Fig. S4 and D). Simultaneous Recording of te Voltage and Firing Rate Rytms. To verify te syncronization of te voltage rytms in te SN, we performed simultaneous recordings of and neuronal firing using a MED wit 8 8 planar electrodes (eac, μm) (Fig. 5). Te cultured SN slice transfected wit AAV encoding SynI was flipped over and placed on te MED probe (Fig. S1, Protocol 2). signals were measured by a igly sensitive D camera mounted on an uprigt microscope, and multiunit spontaneous firings were measured by te MED system. Te voltage and firing rytms were analyzed in μm regions of interest (ROIs) on eac MED electrode. A typical example is sown in Fig. 5D. Te distribution of acropase on pixel or ROI level was unimodal in te respective voltage and firing rytm, indicating syncronization of te circadian rytms trougout te SN (Fig. 5 and ). We analyzed te pase relationsip between te firing and voltage rytms in 39 electrodes (tree slices) and found tat te firing rytms were significantly paseadvanced (by 3.9 ± 0.3 ) relative to te voltage rytms (Fig. 5E, Lower). Tere was no significant regional difference in pase difference between te dorsal (4.1 ± 0.5 ) and ventral (3.8 ± 0.4 ) SN regions (P = 0.644) (Fig. 5F). Togeter, tese results indicate tat te voltage and firing rytms are syncronous trougout te entire SN network and are coupled to eac oter wit a pase difference of 4. E Enoki et al.

6 D E NEUROSIENE PNAS PLUS A F Fig. 5. Spatiotemporal profiles of te firing and voltage rytms. (A) Images of fluorescence (Left) and of MED in brigtfield (Rigt). () An acropase map of te voltage rytm (Upper) and a pase distribution istogram (Lower). () Acropase of te firing rytm (Upper) mapped on eac electrode covering te rigt SN and te pase distribution istogram (Lower). (D) Representative circadian voltage rytms (green traces) and firing rytms (red traces). Data were detrended by 24 moving average subtraction and were normalized relative to te peak amplitude. (E) Histograms of te acropase of firing rytm (Upper) and te pase difference (ΔPase) between te two rytms (Lower) examined in te ROIs covering eac electrode (tree slices, 39 electrodes). Te colored columns represent tree individual experiments. (F) Te pase difference (ΔPase) between te voltage and firing rytms in te dorsal and ventral regions. All data are given as te mean ± SEM; n.s., statistically not significant. Voltage and a2+ Rytms During Development. Previously, we ob served tat 10% of MED electrodes demonstrated firing rytms tat were antipasic to te majority rytms (38, 39). However, te firing rytms were syncronous in all MED electrodes in te present study. ecause te SN slices in tis study were cultured for longer periods of time tan in previous studies, we tested te possibility of developmental canges in te SN network for syncronization. To tis end, we performed simultaneous recordings of te voltage and a2+ rytms for more tan 10 d starting at an early developmental stage (Fig. 6). Te SN slice was prepared from mice at day P1. Entire SN neurons were transfected wit two AAVs, one encoding and te oter, on days P2 and P3, and confocal recordings were started 1 wk later, on corresponding (cp) day 10 (Fig. S1, Protocol 2). We Enoki et al. found tat te topological patterns of te voltage and a2+ rytms in te SN were similar in bot te early and late recording stages (Fig. 6A) (n = 6 slices). However, judging from te SD, te voltage rytms were more syncronized at te late stage (SD = 3.0 ± 0.1 ) (days cp21 22) tan at te early stage (SD = 4.0 ± 0.3 ) (days cp10 11) (P = 0.038) (Fig. 6). y contrast, te a2+ rytms were not significantly different between te early (4.7 ± 0.1 ) and late (4.8 ± 0.03 ) stages (P = 0.87) (Fig. 6). We calculated te pase difference between te voltage and a2+ rytms separately in te dorsal (3.2 ± 0.2 vs. 3.9 ± 0.5 ) and ventral SN ( 1.3 ± 0.6 vs. 0.9 ± 0.4 ) at bot te early and late stage and found no significant regional difference between te two stages (Fig. 6D). Tese results indicate tat te coerence of voltage rytms, but not of te a2+ rytms, is developmentally regulated in te SN. PNAS Publised online Marc 7, 2017 E2481

7 A cp days 10 t 11 t cp days 21 st 22 nd Dorsal orresponding Postnatal Days cp days 10 t 11 t * cp days 21 st 22 nd n.s Early Late Early Late D Pase () () 6 Ventral Vm detrended time () orresponding Postnatal Days Vm detrended time () Early Pase () () 2 Dorsal Ventral Dorsal Ventral Late Fig. 6. Longterm recording of te voltage and rytms. (A) Acropase maps of te voltage rytm (Upper) and rytm (Lower) at te early stage (days cp10 cp11) (Left) and te late stage (days cp21 cp22) (Rigt) of longterm recording. Te mean acropase of te entire SN regions was separately normalized to zero for voltage and rytms. () Time course of te voltage rytms (green traces) and rytms (red traces) in te dorsal (Upper) and ventral (Lower) regions during 13 d of recording. () Network syncronization of te voltage (Left) and (Rigt) rytms at te early and late stages of te recordings. Te variability of network syncrony is represented by te acropase SD. (D) Pase difference (ΔPase) between voltage and rytms in te dorsal and ventral SN at te early (Left) and late (Rigt) stages of te recording (n = 5). *P < Data are given as te mean ± SEM. Discussion In te present study, we found celltype specific couplings of circadian rytms, te syncronous circadian voltage rytms as well as te firing rytms in te entire SN, despite topologically specific rytms. We also found tat ion cannel blockers ad differential effects on te amplitude of voltage and rytms in te AVP neurons but not in te VIP neurons. Tese findings are explained by assuming an interaction between an intrinsic cellular oscillation and te inputs from te SN neural network. elltype Specificity of te ircadian Functions. ircadian rytms in te expression of clock genes, suc as Per1 and Per2, ave been reported to ave topological patterns similar to tose of rytms(6,7);tusterytmsin and clock gene expression and te rytms in voltage and firing beave similarly (Fig. 6A). Tese results imply tat SN neurons ave two functionally coupled oscillatory components: One is composed of te voltage and neuronal firing rytms, and te oter is composed of and PER2 rytms. Tese two oscillatory components migt interact directly or troug te core molecular loop for circadian oscillation. Te pase relationsip of te circadian PER2 and rytms is not different in VIP and AVP neurons, and te same is true for te circadian voltage and firing rytms. However, te pase relationsip between te former component and te latter was substantially different in te two neurons (Figs. 2D, 3G, and 4G). ecause te voltage rytms were syncronous trougout te entire SN, te cellspecific difference is ascribed to te oscillatory component of circadian and PER2 rytms. Interestingly, canged te pase relationsip between te two components in te AVP neurons (Fig. 4G), suggesting tat te SN neural network is involved in te coupling between tem. did not affect te coupling in te VIP neurons (Fig. 3G). As a result, te pase relationsip of two oscillatory components was canged between te AVP and VIP neurons. Te findings are consistent wit our previous report tat desyncronizes te circadian rytms between te dorsal and ventral regions of te SN (7). Signal Origin of te Voltage Rytm. Te voltage rytms were syncronous trougout te entire SN (Fig. 1). Te finding is unique in te face of regional differences in te circadian pase of oter measures suc as clock gene expression in te SN (3). In tis study, we used pixellevel analysis using timeseries D images (7, 30, 40). Eac pixel size is smaller tan te average size of te cell body of a single SN neuron. We are not able to exclude te possibility tat some pixels represent te soma of a certain cell and oter pixels represent te processes of different cells. However, suc compartmentalization cannot explain te syncrony of te voltage rytms trougout te entire SN (Fig. 2). We sowed te syncronization of te voltage rytms in te same group of SN neurons (Figs. 3 and 4), including te regions were te somas and processes exist intermixed (Fig. 1 and and Fig. S2). Te dendrite is known as te major target of synaptic inputs in a variety of neurons. Te voltage canges in te dendrites propagate in te process and soma witin a few second, based on te cable properties of te dendrite (41, 42). Te syncrony of E Enoki et al.

8 voltage rytm trougout te SN migt be caused by a rapid propagation of a subcellular voltage cange. Neural Network and Intracellular ouplings of ircadian Rytms. Application of te cannel blockers for eiter fast Na + or Ltype cannels reduced te amplitude of bot te and voltage rytms in te VIP neurons but only diminised te amplitude of te rytm in te AVP neurons. Te amplitude of te circadian voltage rytm in te AVP neurons persisted witout damping. Te neuronspecific differences suggest te impairment of a specific patway from te intrinsic circadian oscillation to bot overt rytms and/or desyncrony among cellular rytms rater tan a loss of cellintrinsic rytms in particular types of neurons. In addition, a significant cange in te pase difference between te and voltage rytms indicates desyncrony between tem and suggests tat te two circadian rytms are regulated by different mecanisms or at least troug different patways from te core molecular loop of circadian oscillation in te AVP neurons. According to te teory of a multioscillator system (43), te pase of an intrinsic cellular circadian oscillation is affected by but is not completely in pase wit te system oscillation in wic te cell involved. Te circadian voltage and firing rytms may represent te system oscillation in te SN, and te circadian and PER2 rytms may represent te intrinsic cellular oscillation. Interruption of te inputs from te neural system by may release te intrinsic oscillation from te impact of system oscillation to cange te caracteristics of cellular oscillation. Te present findings are adequately explained by tis teory. Possible Mecanisms of Intra and Intercellular ouplings. ased on te present findings, we advanced a model in wic te celltype specificity of circadian rytms is explained by asymmetric impacts from te VIP to AVP neurons. In tis model, te SN neural network differentially influences te two oscillatory cell components in wic te voltage and rytms are involved separately (Fig. 7). Te notion is based on te differential effects of and nimodipine on te amplitude of voltage rytms in te two neuron groups (Figs. 3F and 4F). Te neural network sensitive to or nimodipine as a muc greater influence on te rytms tan on te voltage rytms in te AVP neurons. In te SN, neuronal firings and conductance contribute te membrane potential (34, 36). blocks fast Na + cannels and neuronal firings, and it also suppresses te neuronal inputs from network (Fig. 7, 1). Nimodipine inibits Ltype cannels, tus suppressing and voltage rytms in te AVP and VIP neurons (Fig. 7, 2). Inibition of eiter fast Na + or Ltype cannels enances te amplitude of te voltage rytm in AVP neurons, probably by disinibiting te coupling from te VIP to AVP neurons (Fig. 7, 3). Alternatively, oter ion cannels, suc as te calciumactivated K potassium cannel (5, 44, 45), wic is more abundant in te AVP neurons tan in te VIP neurons, migt be involved. Suppression of rytm amplitude may weaken te coupling between te two oscillatory components, tereby canging te pase relationsip of te rytms. Anoter possible explanation is a cange in te tresold for generating neuronal firings by modulating te ion cannel activation or inactivation. Te firing rytms paselead te voltage rytms by nearly 4. Tis lead is not predictable from te experiments in molluscan clock neurons (46) or te Hodgkin Huxley model in neurons. Te maximum level of depolarization was associated wit a decline in te firing rate. Depolarization block is a possible explanation of tis pase difference but is unlikely, considering tat te estimated amplitude of te voltage rytm was as small as 6.3 mv on average (Fig. S3). Unknown circadian mecanisms, suc as activation of a calciumactivated Ktype potassium cannel and a cange in action potential tresold migt account for tis penomenon. Developmental Regulation of te Voltage Rytms. We found tat te voltage rytms became coerent during te course of recordings (Fig. 6), possibly as te result of te maturation of te SN network during te development. To support tis idea, we recently found VIP and AVP signaling differentially integrated in te SN neural network during development (40). It is wort noting tat te amplitude of te voltage and rytms became larger during culturing. Rytm amplitude could be reinforced by intercellular coupling during development. oerent and robust voltage rytms would lead to stable output signals NEUROSIENE PNAS PLUS A firing voltage PER2 Dorsal Ventral Acropase AVP neuron firing voltage PER2 VIP neuron Pase () AVP neuron Nimodipine VIP neuron PER2 + voltage Nimodipine PER voltage firing firing coupling suppress enance Fig. 7. Summary scema of te spatiotemporal profiles and celltype specificity of te circadian rytms. (A) Spatial patterns of te circadian firing, voltage,, and PER2 rytms. Acropases are scematically sown in pseudocolor wit te mean pase of te entire slice set to 12. () Estimated temporal orders of te circadian rytms in te AVP (Upper) and VIP (Lower) neurons. () Scematic drawing for te functional links between te two oscillating cell components and between te VIP and AVP neurons. Nerve terminals on te rigt represent inputs from te SN networks. Doubleeaded arrows indicate te stable pase relationsip of te two functions suggesting strong coupling. Arrows indicate te direction of te effect. Plus and minus signs denote enancement and suppression effects, respectively, and numbers witin circles (1 3) sow te effects of blockers (see Discussion for details). Enoki et al. PNAS Publised online Marc 7, 2017 E2483

9 and ultimately would sustain coerent and robust rytms in animal beaviors. Te function is of special importance in maintaining activity rest cyclicity under perturbing influences suc as transmeridian fligts and extreme potoperiods. Pysiological Roles of Differentially Pased Voltage and Rytms. Te pysiological roles of te syncronous voltage and asyncronous rytms are unknown, but it is surmised tat te SN uses two rytms differentially for te circadian clockwork: te voltage rytms for uniform sensitivity to environmental stimulation and te rytms for te regionalspecific responses. Previously, in vivo recording using stationary electrodes sowed tat te peak pase of te firing rytms was similar in te dorsal and ventral SN (16). Furtermore, a fixed pase relationsip between Per1 and firing rate as been reported in te SN slices; owever, te spatial pattern and regional specificity were not reported (47). On te oter and, differential responses in te firing and Per1 rytms to a sifted ligt dark cycle ave been reported bot in vivo and in vitro (18). Te pase sift was large and persistent in te Per1 rytm but was transient in te firing rytm in vitro. Te SN network may keep te sensitivity of circadian clock troug te circadian voltage rytm and respond rapidly to environmental stimulation troug te rytms. Furter studies will be required to clarify te pysiological roles of te syncronous voltage rytms in te SN. Materials and Metods Animal are. 57L/6J mice (lea Japan), AVPre mice (12), and Vip tm1(cre)zj /J mice (Jackson Laboratory) on te 57L/6J background were used for te experiments. Mice were born and bred in our animal quarters under controlled environmental conditions (temperature: 22 ± 2, umidity: 60 ± 5%, 12 ligt/12 dark, wit ligts on from 0600 to 1800 ). Ligt intensity was around 100 lx at te cage surface. Te mice were fed commercial cow and tap water ad libitum. Experiments were conducted in compliance wit te rules and regulations establised by te Animal are and Use ommittee of Hokkaido University under te etical permission of te Animal Researc ommittee of Hokkaido University (approval no ). SN Slice ulture. Mice were decapitated in te middle of te ligt pase. Te brains of neonate (1dold or 4 to 6dold) male and female mice were removed rapidly and dipped in an icecold balanced salt solution comprising 87 mm Nal, 2.5 mm Kl, 7 mm Mgl 2, 0.5 mm al 2, 1.25 mm NaH 2 PO 4, 25 mm NaHO 3, 25 mm glucose, 10 mm Hepes, and 75 mm sucrose. A 200μm coronal brain slice containing te mid rostrocaudal region of te SN was carefully prepared using a vibratome (VT 1200; Leica). Te bilateral SNs were dissected from te slice using a surgical knife and were explanted onto a culture membrane (Millicell M; pore size, 0.4 μm; Millipore) in a 35mm Petri dis containing 1.0 ml of DMEM (Invitrogen) and 5% FS (SigmaAldric). efore te recordings, te membrane containing te cultured SN slice was cut out, flipped over, and transferred to glassbottomed dises (35 mm, No. 1S; AG Tecno Glass) or MED probes (20 20 μm) tat were collagen coated (ellmatrix type 1, Nitta Gelatin; Wako) and supplemented wit μl DMEM containing 5% supplement solution. MED dises were sealed wit O 2 permeable filters (HigSensitivity Membrane Kit; YSI) using silicone grease compounds (SH111; Dow orning Toray). AAVMediated Gene Transfer into SN Slices. Aliquots of te AAV (1 μl) arboring Syn1j1 (37), Syn1, Syn1Flex (produced by te University of Pennsylvania Gene Terapy Program Vector ore), and custommade Elongation Factor 1 (EF1) α were inoculated onto te surface of te SN cultures on day 4 6 of culture (Protocol 1) or on day 2 of culture (Protocol 2) (Fig. S1). Wen multiple sensors were transfected in te SN, AAVs were transfected on two subsequent days. Infected slices were cultured furter for at least 14 d before confocal imaging (Protocol 1) or for 7 d before D/MED imaging (Protocol 2) (Fig. S1). Te titers of Syn1j1, Syn1, and Syn1Flex vector were , , and genome copies/ml, respectively. To make AAV encoding EF1α, te AAV2 inverted terminal repeat (ITR) containing plasmid paavef1αdior2(h134r)eyfp (provided by K. Deisserot, Stanford University, Stanford, A) was modified to construct paavef1α 1 by replacing DIOR2(H134R)EYFP cdna wit te 1 cdna fragment from te plasmid MV1 (provided by T. Nagai, Osaka University, Osaka). AAV wit a mutant form of te AAV2 cap gene (provided by A. Srivastava, University of Florida, Gainesville, FL) (48) was produced using a tripletransfection, elperfree metod and was purified as described previously (49). Te titer of vector was genome copies/ml. HigResolution onfocal Imaging of and. Fluorescence images were captured at an exposure of 2 5 s. Images of 100μm dept in te z axis were obtained at 2μm zsteps. Te imaging system was composed of a Nipkow spinning disk confocal microscope (XLigt; rest Optics), a NEO smos D camera (2,560 2,160 pixels, 0.325μm resolution) (Andor Tecnology) or an ixon3 EMD camera (1,024 1,024 pixels, μm resolution) (Andor Tecnology), a TiE inverted microscope (Nikon), Plan Apo V dry objectives (20, 0.75 NA) (Nikon), a TIXH box incubator (Tokaiit), and MetaMorp software (Molecular Devices). was excited by cyan color (475/28 nm) wit an LED ligt source (Spectra X Ligt Engine; Lumencor, Inc.), and te fluorescence was visualized wit 495nm dicroic mirror and 550/49nm emission filters (Semrock). /j was excited by green color (542/27 nm), and te fluorescence was visualized wit 593nm dicroic mirror and 630/92nm emission filters (Semrock). Expression patterns of fluorescence were visualized using confocal laserscanning microscopy (1,024 1,024 pixels) (A1RFN1; Nikon). All experiments were performed at 36.5 and 5% O 2. Simultaneous Recording of te and Spontaneous Firing. Te SN slice was cultured in 100% air at 36.5 on an MED probe wit 64 electrodes (20 20 μm) arranged in an 8 8 grid wit a distance of 100 μm between electrodes. efore te recording, te MED dis was placed in a miniincubator installed on te stage of a microscope (Eclipse 80i; Nikon). fluorescence and spontaneous firing were recorded simultaneously wile culturing in culture medium. Fluorescence was recorded wit a cooled D camera at 80 (ImagEM; Hamamatsu Potonics) every 60 min wit an exposure time of 2 3 s. was excited wit an LED ligt (Ligt Engine; Lumencor, Inc.) at cyan color (475/28 nm), and te fluorescence was visualized wit 495nm dicroic mirrors and 520/35nm emission filters (Semrock). Patclamp Recordings. Wolecell patcclamp recordings were made from cultured SN slice neurons. Neurons were visualized wit a 60 waterimmersion objective lens (LumPlanFL N, 1.0 NA; Olympus) using an uprigt microscope (X50WI; Olympus) equipped wit infrared/differential interference contrast systems and an EMD camera (ImagEM; Hamamatsu Potonics) and a spinning disk confocal unit (SU10; Yokogawa Electric). Te wolecell electrodes (resistance of 5 7 MΩ) were fabricated from borosilicate capillaries (GD1.5; Narisige Scientific Instruments) and were pulled on a micropipette puller (Sutter Instrument). Wolecell current clamp recordings were made wit an internal solution containing te following: 140 mm Kgluconate, 4 mm Kl, 0.2 mm Mgl 2, 10 mm Hepes, 0.2 mm EGTA, 2 mm MgATP, 0.2 mm NaGTP, adjusted to ph 7.3 wit KOH. Slices were continuously superfused wit a pysiological recording solution containing te following: 0.81 mm MgSO 4, 5.36 mm Kl, 0.44 mm KH 2 PO 4, 1.26 mm al 2, mm Nal, 0.34 mm Na 2 HPO 4,4.17mMNaHO 3, and 5.55 mm Dglucose, at flow rates of 2 3 ml/min. All experiments were performed at 32. Responses were recorded using a Multilamp700 amplifier, Digidata 1550A, and plamp10.5 (Molecular Devices), filtered at 10 khz, and digitized at 10 khz. For validation of signals, currentclamp recordings were obtained in solutions containing 0.5, 3, 5.36, and 10 mm Kl. Mean membrane potentials were calculated using 1s data from individual cells. Immunoistocemistry. Te SN slices expressing were incubated in DMEM containing 50 μg/ml colcicine for 24 and were fixed wit 4% paraformaldeyde in 0.1 M PS for 60 min at room temperature. Nonspecific antibody binding was blocked by 60min incubation wit skim milk at room temperature. For labeling AVP and VIP neurons, te SN slices were stained using mouse anti AVP monoclonal antibody (generous gift of H. Gainer, NIH/National Institute of Neurological Disorders and Stroke, etesda) (1:1,000 dilution) and rabbit antivip polyclonal antibody (1:10,000 dilution; Peptide Institute), respectively. Two days later, Alexa 594conjugated goat IgG (1:200 dilution; Invitrogen) was used as te secondary antibody for mouse IgG (AVP) and rabbit IgG (VIP). Te slices were mounted on a glassbottomed dis wit Prolong GoldDAPI (Invitrogen). Fluorescence was visualized using an ixon3 Nipkow spinning disk confocal and EM D camera (Andor Tecnology) and MetaMorp software (Molecular Devices). Data Analysis and Statistics. Statistical analyses were performed using Prism GrapPad (GrapPad Software). Te group mean was presented as te mean ± SEM. Te t test was used wen two independent group means were compared, and te Mann Witney u test or Welc s t test was used wen E Enoki et al.

10 te variances of two group means were different. A paired t test or a onesample t test was used wen two dependent group means were compared. Peak pases of te rytms were estimated by te midpoint of te rising and falling limbs of detrended circadian rytm tat intersected te x axis. For regional comparison of te rytms, μm ROIs in te upper tird (near te tird ventricle) and in te bottom twotirds (near te optic ciasma) were selected as te dorsal and ventral regions. To quantify te rytms at te SN network, we used a custommade program for te creation of acropase maps as described previously (7, 30, 31). riefly, fluorescence images were smooted wit te median filter (one pixel) and converted to eigtbit intensity. ackground signals were selected from a region were no cells were found (referred to as te background region ). Te mean + 5 SDs of te signal intensity of te background region was set as te cutoff level of te signal and background. Te time series of te images in eac pixel [Yj (ti); ti = 1, 2,..., N ()] was fitted to cosine curve yj(t) = yj (t;mj,aj,j,tj)= Mj + Aj*cosine[2pi(t j)/tj] using a leastsquare regression metod, were yj(t) is te signal intensity at time t (), Mj is te mesor, Aj is te amplitude, j is te acropase, and Tj is te period of te images. Te goodness of fit was statistically evaluated by te percent of rytm accounted for by te fitted cosine wave (Pearson productmoment correlation analysis) at a significance level of P < In all figures, acropase maps are sown by pseudocolor, and pixels wit unfitted rytms and background level signals are sown 1. Moore RY, Eicler V (1972) Loss of a circadian adrenal corticosterone rytm following supraciasmatic lesions in te rat. rain Res 42(1): Moawk JA, Green, Takaasi JS (2012) entral and periperal circadian clocks in mammals. Annu Rev Neurosci 35(1): Wels DK, Takaasi JS, Kay SA (2010) Supraciasmatic nucleus: ell autonomy and network properties. Annu Rev Pysiol 72: Reppert SM, Weaver DR (2002) oordination of circadian timing in mammals. Nature 418(6901): olwell S (2011) Linking neural activity and molecular oscillations in te SN. Nat Rev Neurosci 12(10): Yamaguci S, et al. (2003) Syncronization of cellular clocks in te supraciasmatic nucleus. Science 302(5649): Enoki R, et al. (2012) Topological specificity and ierarcical network of te circadian calcium rytm in te supraciasmatic nucleus. Proc Natl Acad Sci USA 109(52): Scwartz WJ, Gross RA, Morton MT (1987) Te supraciasmatic nuclei contain a tetrodotoxinresistant circadian pacemaker. Proc Natl Acad Sci USA 84(6): rancaccio M, Maywood ES, esam JE, Loudon AS, Hastings MH (2013) A Gq axis controls circuitlevel encoding of circadian time in te supraciasmatic nucleus. Neuron 78(4): Inagaki N, Honma S, Ono D, Tanaasi Y, Honma K (2007) Separate oscillating cell groups in mouse supraciasmatic nucleus couple potoperiodically to te onset and end of daily activity. Proc Natl Acad Sci USA 104(18): Farajnia S, van Westering TLE, Meijer JH, Micel S (2014) Seasonal induction of GAAergic excitation in te central mammalian clock. Proc Natl Acad Sci USA 111(26): Mieda M, et al. (2015) ellular clocks in AVP neurons of te SN are critical for interneuronal coupling regulating circadian beavior rytm. Neuron 85(5): Nagano M, et al. (2003) An abrupt sift in te day/nigt cycle causes desyncrony in te mammalian circadian center. J Neurosci 23(14): Evans JA, Leise TL, astanonervantes O, Davidson AJ (2013) Dynamic interactions mediated by nonredundant signaling mecanisms couple circadian clock neurons. Neuron 80(4): rancaccio M, et al. (2014) Networkmediated encoding of circadian time: Te supraciasmatic nucleus (SN) from genes to neurons to circuits, and back. J Neurosci 34(46): Scaap J, et al. (2003) Heterogeneity of rytmic supraciasmatic nucleus neurons: Implications for circadian waveform and potoperiodic encoding. Proc Natl Acad Sci USA 100(26): Albus H, Vansteensel MJ, Micel S, lock GD, Meijer JH (2005) A GAAergic mecanism is necessary for coupling dissociable ventral and dorsal regional oscillators witin te circadian clock. urr iol 15(10): Vansteensel MJ, et al. (2003) Dissociation between circadian Per1 and neuronal and beavioral rytms following a sifted environmental cycle. urr iol 13(17): VanderLeest HT, et al. (2007) Seasonal encoding by te circadian pacemaker of te SN. urr iol 17(5): Sirakawa T, Honma S, Katsuno Y, Oguci H, Honma KI (2000) Syncronization of circadian firing rytms in cultured rat supraciasmatic neurons. Eur J Neurosci 12(8): en TW, et al. (2013) Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499(7458): uzsáki G, Dragun A (2004) Neuronal oscillations in cortical networks. Science 304(5679): Marder E, Abbott LF, Turrigiano GG, Liu Z, Golowasc J (1996) Memory from te dynamics of intrinsic membrane currents. Proc Natl Acad Sci USA 93(24): elle MD, Diekman O, Forger D, Piggins HD (2009) Daily electrical silencing in te mammalian circadian clock. Science 326(5950): by wite color. Te mean acropase of te entire SN regions is separately normalized to zero for voltage and rytms. To validate te rytmicity in te ROIs, fluorescence signals in eac ROI were fitted to cosine curves using a leastsquare regression metod, and te goodness of fit was evaluated by cosine curve fitting and percent rytm (P < 0.001). AKNOWLEDGMENTS. We tank te Genetically Encoded Neuronal Indicator and Effector (GENIE) Project and te Janelia Farm Researc ampus of te Howard Huges Medical Institute for saring j1 constructs; Dr. H. Gainer (NIH/National Institute of Neurological Disorders and Stroke) for providing antiarginine vasopressin monoclonal antibody; Dr. T. Nagai (Osaka University) for supplying plasmid; and Dr. S. Kuroda (Hokkaido University) for providing te analysis program. Tis work was supported by Ministry of Education, ulture, Sports, Science and Tecnology (MEXT)/Japan Society for te Promotion of Science Researc Grants , , and 15KT0072 (to R.E.), and 15H04679 (to S.H.), 16K16645 (to Y.O.), and 16H05120 (to M.M.); te Precursory Researc for Embryonic Science and Tecnology/Japan Science and Tecnology Agency; te ooperative Researc Project for Advanced Potonic ioimaging; te Project for Developing Innovation Systems of MEXT; Akiyama Life Science Foundation (R.E.), te Japan Foundation for Neuroscience and Mental Healt (R.E.), and te Narisige Neuroscience Researc Foundation (R.E.). 25. Ross WN, et al. (1977) anges in absorption, fluorescence, dicroism, and irefringence in stained giant axons: Optical measurement of membrane potential. J Membr iol 33(1 2): Nakajima R, Jung A, Yoon J, aker J (2016) Optogenetic monitoring of synaptic activity wit genetically encoded voltage indicators. Front Synaptic Neurosci 8(August): Jin L, et al. (2012) Single action potentials and subtresold electrical events imaged in neurons wit a fluorescent protein voltage probe. Neuron 75(5): Enoki R, Ono D, Hasan MT, Honma S, Honma K (2012) Singlecell resolution fluorescence imaging of circadian rytms detected wit a Nipkow spinning disk confocal system. J Neurosci Metods 207(1): Zao Y, et al. (2011) An expanded palette of genetically encoded indicators. Science 333(6051): Yosikawa T, et al. (2015) Spatiotemporal profiles of arginine vasopressin transcription in cultured supraciasmatic nucleus. Eur J Neurosci 42(9): Honma S, et al. (2016) Oscillator networks in te supraciasmatic nucleus: Analysis of circadian parameters using timelaps images. iological locks, eds Honma K, Honma S (Hokkaido Univ Press, Sapporo, Japan), pp Lundkvist G, Kwak Y, Davis EK, Tei H, lock GD (2005) A calcium flux is required for circadian rytm generation in mammalian pacemaker neurons. J Neurosci 25(33): Ikeda M, et al. (2003) ircadian dynamics of cytosolic and nuclear in single supraciasmatic nucleus neurons. Neuron 38(2): Irwin RP, Allen N (2007) alcium response to retinoypotalamic tract synaptic transmission in supraciasmatic nucleus neurons. J Neurosci 27(43): Pennartz M, de Jeu MT, os NP, Scaap J, Geurtsen AM (2002) Diurnal modulation of pacemaker potentials and calcium current in te mammalian circadian clock. Nature 416(6878): Diekman O, et al. (2013) auses and consequences of yperexcitation in central clock neurons. PLOS omput iol 9(8):e Dana H, et al. (2016) Sensitive red protein calcium indicators for imaging neural activity. elife 5: Ono D, Honma S, Honma K (2013) ryptocromes are critical for te development of coerent circadian rytms in te mouse supraciasmatic nucleus. Nat ommun 4: Enoki R, Ono D, Kuroda S, Honma S, Honma KI (2017) Dual origins of te intracellular circadian calcium rytm in te supraciasmatic nucleus. Sci Rep 7: Ono D, Honma S, Honma K (2016) Differential roles of AVP and VIP signaling in te postnatal canges of neural networks for coerent circadian rytms in te SN. Sci Adv 2(9):e Enoki R, Namiki M, Kudo Y, Miyakawa H (2002) Optical monitoring of synaptic summation along te dendrites of A1 pyramidal neurons. Neuroscience 113(4): Kasuga A, et al. (2003) Optical detection of dendritic spike initiation in ippocampal A1 pyramidal neurons. Neuroscience 118(4): Tokuda IT, et al. (2015) oupling controls te syncrony of clock cells in development and knockouts. iopys J 109(10): Kent J, Meredit AL (2008) K cannels regulate spontaneous action potential rytmicity in te supraciasmatic nucleus. PLoS One 3(12):e3884, /journal. pone Meredit AL, et al. (2006) K calciumactivated potassium cannels regulate circadian beavioral rytms and pacemaker output. Nat Neurosci 9(8): lock GD, McMaon DG (1984) ellular analysis of te ulla ocular circadian pacemaker system. J omp Pysiol A Neuroetol Sens Neural eav Pysiol 155(3): Jones JR, McMaon DG (2016) Te core clock gene Per1 pases molecular and electrical circadian rytms in SN neurons. PeerJ 4:e Zong L, et al. (2008) Next generation of adenoassociated virus 2 vectors: Point mutations in tyrosines lead to igefficiency transduction at lower doses. Proc Natl Acad Sci USA 105(22): Hasegawa E, Yanagisawa M, Sakurai T, Mieda M (2014) Orexin neurons suppress narcolepsy via 2 distinct efferent patways. J lin Invest 124(2): NEUROSIENE PNAS PLUS Enoki et al. PNAS Publised online Marc 7, 2017 E2485