Neuronal feedback loop of the suprachiasmatic nucleus generates robust circadian rhythms

Domain: neuroscience
Published: 2026-01-05
Editorial outcome: accept

Abstract
The central circadian clock of the suprachiasmatic nucleus (SCN) comprises a network of diverse neuronal and glial cell types, yet its operating mechanism remains elusive. Here, by monitoring cellular calcium rhythms in vivo using dual-color fiber photometry in mice lacking vasoactive intestinal polypeptide (VIP), we demonstrate that arginine vasopressin (AVP) neurons oscillate intrinsically with a short period and reduced amplitude. This indicates that VIP normally amplifies and phase-delays the AVP neuronal rhythm each day, thereby lengthening its period to approximately 24 h in constant darkness. Consistently, the behavioral circadian period is shortened by AVP neuron-specific VIP receptor dysfunction and lengthened by AVP neuron-specific blockade of neurotransmitter release. VIP neurons and other SCN cell types occasionally exhibit weak, unstable, long-period calcium rhythms only when the AVP neuronal oscillation is attenuated due to VIP deficiency or AVP neuron-specific Bmal1 deletion. Given that AVP neurons serve as the primary pacesetter cells of the SCN ensemble rhythm, these results indicate that an SCN neuronal feedback loop (SNFL), composed of the AVP cellular oscillator and VIPergic signaling, is essential for generating robust circadian rhythms.

Introduction
The suprachiasmatic nucleus (SCN) acts as the central circadian pacemaker in mammals, orchestrating multiple circadian rhythms of behavior and physiological function according to the environmental light/dark (LD) cues conveyed from the eye1–4. The SCN contains ~20,000 cells, most of which can generate circadian oscillations. In individual cells, the circadian rhythm is driven by the autoregulatory transcription-translation feedback loop (TTFL) consisting of clock genes such as Per1/2/3, Cry1/2, Bmal1, and Clock1,2. Notably, the TTFL is not unique to SCN cells and is common to almost all peripheral cells2,5. Rather, the SCN constructs a network of many TTFL-driven cellular clocks to generate a highly robust and coherent circadian rhythm as the central clock, which entrains a large number of peripheral clocks throughout the body1–4. Thus, intercellular communication among SCN clock cells through the neuronal and diffusible network distinguishes the SCN from the peripheral clocks. However, the network mechanism underlying the SCN central clock remains unclear.

The SCN is a heterogeneous structure composed of multiple types of neurons and glial cells1–4. Most SCN neurons are GABAergic, and several populations of these GABAergic neurons also co-express neuropeptides. These include arginine vasopressin (AVP)-producing neurons located predominantly in the dorsomedial part (SCN shell) and vasoactive intestinal polypeptide (VIP)-producing neurons in the ventrolateral part (SCN core). VIP is the most important contributor to the maintenance and synchrony of SCN cellular clocks with varying periods6,7. Therefore, mice deficient in VIP (Vip−/−) or its receptor VPAC2 (Vipr2−/−) demonstrate a drastic attenuation of the free-running rhythm in constant darkness (DD) with a shortened period or unstable multiple periods. In the latter, the free-running period changes suddenly or two periods emerge simultaneously6,8–10. VIP neurons receive direct projections from the retina, are involved in the photoentrainment, and send dense projections dorsally to the SCN shell1,9,11. However, the precise mechanisms by which VIP regulates SCN circuit remain incompletely understood.

On the other hand, our studies and those of others have suggested that the TTFL-driven cellular clocks in AVP neurons may play a dominant role than those of other SCN neurons in the generation of circadian oscillation by the SCN network, regulating the circadian oscillation of other cells and setting the SCN ensemble period3. Thus, AVP neuron-specific disruption of the TTFL by deleting Bmal1 (Avp-Bmal1−/− mice) results in an unstable, attenuated free-running rhythm with a lengthened period and the activity time (the interval between the onset and offset of locomotor activity)12–16. Furthermore, the cellular period length of AVP neurons appears to be the primary determinant of the SCN ensemble period16,17. In contrast, similar genetic manipulations of disrupting the TTFL or altering the cellular period in VIP neurons have little effect on the amplitude and period of the free-running behavior rhythm13–16,18, suggesting that VIP peptide is critical but the TTFL in VIP neurons may be dispensable for the circadian timekeeping. Besides, our previous studies indicated the importance of in vivo analysis of SCN network dynamics16,17.

Thus, there is a functional differentiation between the shell and the core within the SCN network. However, how the two integrate to generate the circadian rhythm of the SCN ensemble is still unclear. In the current study, we investigated the regulation of AVP neuronal circadian oscillation by VIP in vivo. To this end, we applied dual-color fiber photometry recording to VIP-deficient mice, allowing us to simultaneously monitor the cellular circadian rhythms from two types of SCN neurons. We then analyzed the circadian behavior rhythms of multiple mouse strains that had various impairments in the communication between VIP and AVP neurons. Our results reveal a feedback loop neuronal circuit of these two neuron types that may be the essential network mechanism of the SCN central circadian pacemaker.

Results
AVP neurons maintain a short-period Ca2+ rhythm in VIP-deficient mice
To elucidate the mechanism of how VIP regulates the cellular clocks of AVP neurons, we first aimed to compare the circadian rhythm of intracellular Ca2+ in SCN AVP neurons between control and VIP-deficient mice. Intracellular Ca2+ rhythms are a nice measure of cellular circadian rhythms in vivo that can be recorded in a neuron type-specific manner11,16,19–21. We used the homozygous Vip-tTA knock-in mice (ViptTA/tTA), which are equivalent to Vip knock-out mice and exhibit a spectrum of phenotypes such as simple short-period or complicated multiple-period behavioral rhythms10. In addition, their specific expression of tTA in VIP neurons allowed us to record Ca2+ rhythms in SCN AVP and VIP neurons (AVP- and VIP-Ca2+) separately and simultaneously when combined with hemizygous Avp-Cre BAC transgenic mice12. Thus, we generated VIP-deficient Avp-Cre; ViptTA/tTA mice and heterozygous control Avp-Cre; Vipwt/tTA mice and then targeted the expression of green (jGCaMP7s)22 and red (jRGECO1a)23 fluorescent Ca2+ sensor proteins in AVP and VIP neurons, respectively, by simultaneous injection of Cre- and tTA-dependent AAV vectors (AAV-CAG-FLEX-jGCaMP7s and AAV-TRE-jRGECO1a) (Fig. 1a, b).

In vivo dual-color fiber photometry with simultaneous monitoring of spontaneous locomotor activity revealed robust, temporally coordinated AVP- and VIP-Ca2+ rhythms in control mice in both light/dark (LD) and dark/dark (DD), higher during the (subjective) day (Fig. 1c–e and Supplementary Fig. 1a–c). In VIP-deficient mice, the VIP-Ca2+ rhythm was comparable to that of controls in LD but rapidly attenuated in DD, even when the locomotor activity still showed rhythmicity (Fig. 1c, e and Supplementary Fig. 2). This made it difficult to determine the daily onset and offset of the VIP-Ca2+ rhythm from its waveforms after a week in DD (Fig. 1e–g). The number of jRGECO1a-expressing cells was comparable between control and VIP-deficient mice (Supplementary Fig. 1e), suggesting that the lack of VIP did not decline the VIP neuronal health. In contrast, the amplitude of the AVP-Ca2+ rhythm was substantially reduced in both LD and DD, but it remained stable in DD (Fig. 1c, d and Supplementary Fig. 2). Consistent with the changes in the behavior rhythm, the AVP-Ca2+ rhythm showed a phase advance in LD (midpoint phase: control, ZT 4.11±0.38; VIP-deficient, ZT 21.62±0.80, p<0.001) and a shortened period in DD (control, ~23.7 h; VIP-deficient, ~22.5 h, p<0.05) (Fig. 1f, g and Supplementary Fig. 1b–d). Notably, the latter was almost identical to the short free-running period of the behavior rhythm (determined based on the locomotor activity onsets), even when behavior changed to a long-period rhythm later in DD (Fig. 1c, g and Supplementary Fig. 2). These results suggest that AVP neurons intrinsically oscillate with a short period and that VIP enhances and delays the oscillation of AVP neurons to make their period closer to 24 h in DD. In turn, the enhanced AVP neuronal rhythm may confer a robust circadian oscillation of VIP neurons, regulating the timing of VIP release. In LD, light may be unable to appropriately entrain and boost the AVP-Ca2+ rhythm in the absence of VIP.

In this study, we used mice of both sexes and analyzed them collectively. However, we confirmed that the results remain fundamentally unchanged when analyzing males and females separately (e.g., Supplementary Fig. 3; also see “Animals” in the “Methods”). The sex of each mouse is disclosed in the Supplementary Figs. and the Source Data file.

VIP-Ca2+ changes to a weak, long-period rhythm in VIP-deficient mice
In VIP-deficient mice, the period length of AVP-Ca2+ rhythm was comparable with the short-period behavior rhythm in DD. As a result, in more than half of VIP-deficient mice, the AVP-Ca2+ rhythm dissociated from the long-period behavior rhythm in the later stage of DD. The VIP-Ca2+ rhythm in actograms initially followed the short-period AVP-Ca2+ and behavior rhythms but gradually disappeared and became very weak and noisy in DD (Fig. 1c–g and Supplementary Figs. 1 and 2). Considering the multiple periodicities of the behavior rhythm in VIP-deficient mice6,8–10, we performed periodogram analyses of Ca2+ rhythms throughout three consecutive weeks in DD to detect weak rhythms with different periods objectively and quantitatively. The VIP-Ca2+ rhythm demonstrated two period peaks in most VIP-deficient mice (Supplementary Fig. 2). Averaging periodograms of all the recorded VIP-deficient mice clarified two peaks in the VIP-Ca2+ rhythm, a primary short period (22.67 h) and a secondary long period (24.33 h), whereas AVP-Ca2+ rhythm showed only one short period (22.50 h) (Fig. 2a; period values are multiple of 1/6 because of 10-min bins of Ca2+ recordings). In contrast, both AVP- and VIP-Ca2+ rhythms of control mice presented a robust single period within the normal range (23.67 h), which matched that of the behavior rhythm (Fig. 2a and Supplementary Fig. 1a). We obtained a similar result by first detecting two period peaks for each mouse and then calculating the mean values for the short and long periods of each mouse (AVP-Ca2+: control, 23.60±0.09 h; VIP-deficient, 22.61±0.07 h; VIP-Ca2+: control, 23.60±0.09 h; VIP-deficient primary, 22.67±0.08 h; secondary, 24.23±0.13 h) (Fig. 2b, c).

To determine the stage of DD at which the secondary long period occurred, we next calculated the time courses of amplitudes (Qp) and periods of AVP-Ca2+, VIP-Ca2+, and behavior rhythms by sequential periodogram analyses with 5-day shifting windows. Although 5 days may be too short to precisely calculate the period length, it may be sufficient to briefly track the time course of changing periods. In VIP-deficient mice, the peak Qp values of the VIP-Ca2+ rhythm decreased continuously during the first 7–8 days (early stage) and then sustained at a relatively low value until the end of the recording (late stage) (Fig. 2e, right). At the early stage, there was no significant difference in the period length between AVP-Ca2+, VIP-Ca2+, and behavior rhythms (Fig. 2d, right). At the late stage, however, the period of the VIP-Ca2+ rhythm was lengthened close to the long period of the behavior rhythm (VIP-Ca2+ vs. Locomotor activity, p>0.99), whereas the AVP-Ca2+ period remained short (AVP-Ca2+ vs. VIP-Ca2+, p<0.01; AVP-Ca2+ vs. Locomotor activity, p<0.001, Fig. 2d, right and Supplementary Fig. 2). In control mice, all periods of Ca2+ and behavior rhythms were stable and consistent (Fig. 2d, e, right and Supplementary Fig. 1a). These results suggest that VIP neurons may be a subordinate slow oscillator and exhibit a weak, long-period rhythm that correlates with the behavior rhythm period in vivo when the AVP cellular rhythm is attenuated by VIP deficiency. However, the causal relationship between these weak VIP-Ca2+ and behavior rhythms remains unknown.

AVP neurons are responsible for the short-period behavior rhythm in VIP-deficiency
As described above, we observed a correlation between the AVP-Ca2+ and the short-period behavior rhythms in VIP-deficient mice (Figs. 1g and 2d). To test for the causal relationship, we measured the behavior rhythm of VIP-deficient mice with an artificially lengthened TTFL period in AVP neurons due to the specific deletion of the casein kinase 1 delta (CK1δ) gene (Avp-Cre; CK1δflox/flox; ViptTA/tTA = Avp-CK1δ−/−; ViptTA/tTA mice).

As reported previously17, control mice (Avp-CK1δ−/−; Vipwt/tTA) exhibited a robust free-running rhythm in DD with a lengthened period (24.70±0.05 h) (Fig. 3 and Supplementary Fig. 4a). In contrast, Avp-CK1δ−/−; ViptTA/tTA mice showed an attenuated behavior rhythm with unstable periods, the mean of which was significantly shorter than that of control Avp-CK1δ−/− mice (24.23±0.11 h) (Fig. 3 and Supplementary Fig. 4b). However, unlike CK1δ-intact VIP-deficient mice (Fig. 1g and Supplementary Fig. 2), they never initiated the free-running rhythm with a short period around 22.6 h (Fig. 3a, b, e and Supplementary Fig. 4b). These results indicate that the AVP cellular period regulates the short-period component of the behavior rhythm in VIP-deficient mice.

Ca2+ rhythm of CCK neurons in VIP-deficient mice rapidly attenuates in DD
Next, we aimed to know how unique the consistently short-period AVP-Ca2+ and the late-onset, weak, long-period VIP-Ca2+ rhythms are. In particular, CCK neurons enriched in the anterior shell region of the SCN were of interest as another candidate for a long-period oscillator, because they reportedly play an important role in the behavior under a long-day photoperiod, and their ablation slightly shortened the behavioral free-running period in DD19.

To test this possibility, we introduced a Cck-ires-Cre allele24 to VIP-deficient (Cckwt/ires-Cre; ViptTA/tTA) and control (Cckwt/ires-Cre; Vipwt/tTA) mice and labeled CCK and VIP neurons with jGCaMP7s and jRGECO1a, respectively, by injecting AAV vectors into the SCN (Fig. 4a, b). Our fiber photometry recording successfully recapitulated the reported CCK-Ca2+ rhythm in control mice, with its peak in the second half of the (subjective) day (Fig. 4c, d and Supplementary Fig. 5a)19. In VIP-deficient mice, the CCK-Ca2+ rhythm was robust in LD but rapidly attenuated in DD, as did VIP-Ca2+ (Fig. 4c, d and Supplementary Fig. 5b). However, in contrast to the VIP-Ca2+, the averaged periodogram of the CCK-Ca2+ rhythm for 3-week recordings in DD showed only one period peak (22.00 h), which was even slightly shorter than that of AVP-Ca2+ (22.50 h) but with a broad distribution of significant period lengths on the long-period side up to ~24.3 h, i.e., the secondary peak of the VIP-Ca2+ period (Fig. 4f and Supplementary Fig. 5b). Sequential periodogram analysis revealed that the CCK-Ca2+ rhythm gradually reduced its amplitude (Qp) and maintained a short period on average, but with a large variability (Fig. 4g, h and Supplementary Fig. 5b). In contrast, in control mice, CCK-Ca2+, VIP-Ca2+, and behavioral periods were stable and consistent (23.50 h) (Fig. 4f–h and Supplementary Fig. 5a). These results suggest that the CCK-Ca2+ rhythm in VIP deficiency is weak and includes more heterogeneous periods in DD. Furthermore, the late-onset, weak, long-period rhythm is unlikely to be unique to VIP neurons. Rather, it may be a more general feature of the multiple types of SCN cells that appear when AVP neuronal rhythm is attenuated due to VIP deficiency.

Such heterogeneity may partly reflect that CCK neurons contain at least two subpopulations, one overlapping with a small subpopulation of AVP neurons25,26. To estimate the overlap of AVP and CCK neurons, we mated Cck-ires-Cre mice with Rosa26-LSL-tdTomato (Ai14) reporter mice to label CCK neurons with tdTomato27. Then, we stained their SCN sections with an anti-AVP antibody. Approximately 30% of tdTomato+ cells were also AVP+, whereas ~20% of AVP+ cells were tdTomato+ (Supplementary Fig. 6). These AVP-positive and -negative CCK neurons may correspond to Cck+/C1ql3+ and Cck+/Bdnf1+ cells, respectively, reported by Wen et al.25. Thus, the CCK-Ca2+ rhythm recorded here by fiber photometry likely derived from a mixture of these two CCK neuron subpopulations.

Ablating VIP neurons in adulthood causes a simple short-period rhythm
VIP-deficient ViptTA/tTA mice free-ran initially with a short period in DD, but about 60% of the mice changed the free-running rhythm to a long period after 1–2 weeks (Supplementary Figs. 2 and 5). This observation was consistent with the previous reports6,8,9. On the other hand, mice in which VIP neurons were ablated in adulthood were reported to retain the wheel running rhythm but with a shortened period (~22.7 h)28. We also performed a similar experiment to measure the locomotor activity (home cage activity) rhythm by injecting an AAV vector expressing activated Caspase 3 in a Cre-dependent manner (AAV-CAG-FLEX-taCasp3-TEVp) bilaterally into the SCN of Vipwt/ires-Cre mice24. We obtained a similar result, i.e., a shortened free-running period and reduced amplitude of the rhythm (22.77±0.09 h vs. 23.67±0.04 h, p<0.001) (Supplementary Fig. 7). We observed an apparent sex difference in the amplitude reduction but not in the shortening of the period in VIP neuron-ablated mice (Supplementary Fig. 7e, f). These results suggest that VIP neurons contribute to circadian timekeeping by lengthening the circadian period and promoting the rhythmicity in adults. Some compensatory mechanisms may cause multiple periods in VIP-deficient mice during development28.

VPAC2 dysfunction in AVP neurons acutely shortens the free-running period
We next investigated the importance of VIP/VPAC2 signaling in AVP neurons for the pacesetting of the SCN network. We used in vivo genome editing to selectively disrupt the Vipr2 gene in AVP neurons. First, we crossed Avp-Cre mice with Rosa26-LSL-SpCas9 mice, which express SpCas9 Cre-dependently29. We then injected AAV vectors expressing gRNAs targeting the Vipr2 (AAV-U6-gVipr2-EF1α-DIO-mCherry, Avp-Vipr2−/− mice) or control gRNAs (AAV-U6-gControl-EF1α-DIO-mCherry) bilaterally into the SCN of Avp-Cre; Rosa26-LSL-SpCas9 mice (Fig. 5a, b). Substantial reduction of VPAC2 expression specific to the SCN shell was confirmed in Avp-Vipr2−/− mice by immunohistochemistry (Fig. 5b, c).

Avp-Vipr2−/− mice free-ran with a period significantly shorter than controls when released into DD (23.21±0.05 h vs. 23.73±0.05 h, p<0.001) (Fig. 5d, f and Supplementary Fig. 8b). Surprisingly, however, after a while, Avp-Vipr2−/− mice suddenly changed their free-running period to the one comparable to controls (23.85±0.03 h vs. 23.71±0.03 h, p=0.27). This observation led us to start recording locomotor activity immediately after the AAV injection in most of the mice examined, with recordings in LD only for 3 days before DD (Supplementary Fig. 8b). Strikingly, all Avp-Vipr2−/− mice exhibited changes in the free-running period around 21 days after AAV injection. Control mice never showed such a sudden change in the free-running period (Fig. 5d, f and Supplementary Fig. 8a). These results suggest that disruption of VIP/VPAC2 signaling in AVP neurons in adulthood acutely shortens the free-running period due to the reduced phase-delaying effect of VIP on the intrinsic short period of AVP neuronal clocks. On the other hand, the period shortening in Avp-Vipr2−/− mice was less than in VIP-deficient and VIP neuron-ablated mice (23.21±0.05 h vs. 22.67±0.08 h and 22.77±0.09 h). Moreover, the shortening was abolished ~3 weeks after the blockade of the VIP→AVP neuron axis. These observations may be due to an incomplete elimination of VPAC2 from AVP neurons. In addition, we speculate that the indirect pathways may exist that mediate the regulation of AVP neurons by VIP neurons and are involved in the compensatory recovery of the standard period in Avp-Vipr2−/− mice.

Disrupting TTFLs in AVP neurons leaves an attenuated, long-period VIP-Ca2+ rhythm
We previously reported that Bmal1 deletion specific to AVP neurons (Avp-Cre; Bmal1flox/flox = Avp-Bmal1−/− mice) leaves an attenuated circadian behavior rhythm with a lengthening of the free-running period and the activity time12. In contrast, mice become completely arrhythmic when Bmal1 is deleted in all SCN neurons using forebrain-specific CamKII-Cre driver mice16,30. These results may be explained by the weak circadian oscillators of SCN non-AVP cells with a long period as a population in vivo, including VIP and CCK neurons as described above. To test this possibility, we recorded AVP-Ca2+ and VIP-Ca2+ rhythms in Avp-Bmal1−/− mice. To do so, we generated Avp-Bmal1−/−; Vipwt/tTA mice and injected AAV vectors into the SCN (Fig. 6a, b). Similar fiber photometry recordings from Avp-Cre (Bmal1wt/wt); Vipwt/tTA mice (Figs. 1 and 2) were regarded as controls. As expected, the AVP-Ca2+ exhibited little circadian rhythmicity (Fig. 6c, d, f and Supplementary Fig. 9b, c) in Avp-Bmal1−/−; Vipwt/tTA mice. Notably, the VIP-Ca2+ retained a rhythm with a long period (24.54±0.16 h) that is consistent with the period of the behavior rhythm (24.47±0.16 h) in DD (Fig. 6e–g and Supplementary Fig. 9b, c). The duration of high VIP-Ca2+ in the subjective day was compressed according to the prolongation of the activity time of the behavior rhythm (Fig. 6c and Supplementary Fig. 9b). Consistent with the previous report12, one out of six Avp-Bmal1−/−; Vipwt/tTA mice demonstrated the multiple period phenotype. In this mouse, the VIP-Ca2+ rhythm was significant but largely attenuated (Supplementary Fig. 9b, mouse #6). These results support the idea that an attenuated oscillation of non-AVP cells with a long period on average manifests in the circadian behavior rhythm when the AVP cellular clock is disrupted.

Blocking neurotransmitter release from AVP neurons lengthens the free-running period
To further explore the evidence that AVP neurons act as the principal oscillator with an intrinsically short period, but its attenuation uncovers a weak, long-period circadian oscillation of non-AVP cells, we next examined the behavioral changes in mice in which the neurotransmitter release from AVP neurons was blocked by AAV-mediated expression of tetanus toxin light chain (Avp-TeNT mice, AAV-EF1α-DIO-GFP::TeNT injected bilaterally into the SCN of Avp-Cre mice) (Fig. 7a, b). Avp-TeNT mice showed a longer free-running period (24.48±0.06 h) in DD than control EGFP-expressing mice (23.28±0.06 h, p<0.001, Fig. 7c–e and Supplementary Fig. 10). These data further confirmed that AVP neurons intrinsically encode a short-period circadian rhythm. The amplitude of the locomotor activity rhythm (periodogram Qp) was not significantly different between groups (p=0.86, Fig. 7d, e). However, individual Avp-TeNT mice exhibited diverse daily patterns of locomotor activity that deviated significantly from the normal nocturnal and bimodal pattern with clear evening (E) and morning (M) locomotor activities (Fig. 7c and Supplementary Fig. 10b). To our surprise, many Avp-TeNT mice showed varying degrees of diurnal locomotor activity rhythms in both LD and DD. To understand the underlying cause, we followed the time course of the emergence of apparent diurnal rhythms in LD after the onset of TeNT expression. Interestingly, TeNT expression in AVP neurons rapidly altered the coupling of E and M locomotor activities (Supplementary Fig. 10c). Namely, Avp-TeNT mice quickly exhibited a prolonged activity time, specifically manifested as a slight advancement in locomotor activity onset (E activity) and a significant delay in locomotor activity offset (M activity). A new steady state of the M-E (M is closer to the following E than to the preceding E) phase relation, rather than the normal E-M phase relation, generated an apparent diurnal behavior rhythm in some mice. Such features can be regarded as a more severe phenotype of the lengthened activity time observed in Avp-Bmal1−/− and Avp-Vgat−/− mice12,20. It should be noted that the free-running period was consistently lengthened in Avp-TeNT mice regardless of their diverse daily patterns of locomotor activity. Thus, these data suggest that blocking neurotransmission of AVP neurons impairs both the pacesetting of the ensemble period and the phase-setting of both E and M locomotor activities.

Neuronal feedback loop model of the SCN explains experimental results
Taken together with previous findings that AVP neurons act as the primary circadian pacesetter in vivo3,12,16,17, the above results suggest a feedback loop neural circuit of AVP and VIP neurons in the SCN (SNFL: SCN neuronal feedback loop) as a critical network machinery of the mammalian central clock (Fig. 8a), which generates robust circadian rhythms with a period of ~24 h in DD. In this context, AVP neurons are the principal oscillatory part of the SCN and regulate the circadian rhythms of VIP and other SCN cells16. AVP neurons constitute a fast oscillator with an intrinsically short period. VIP peptide, released rhythmically, acts on AVP neurons, delaying the AVP neuronal rhythm every day to lengthen its period closer to 24 h and increase its amplitude (Fig. 8a, right). In addition to this primary direct pathway, there may also be minor indirect routes through which VIP regulates AVP neurons via other SCN cells (Fig. 5). Consequently, in the absence of VIP, the AVP neuronal rhythm expresses an intrinsic short period in the attenuated behavior rhythm (Fig. 8a, middle). However, when AVP neuronal oscillation reduces its amplitude too much to control the various clock cells in the SCN, which have different cellular period lengths but an average long period, a weak long-period behavior rhythm emerges (background circadian oscillation or default clock state, discussed below in the Discussion) (Fig. 8a, left). Such rhythms also appear when the pacesetting ability of AVP neurons is impaired by specifically disrupting TTFL or blocking transmitter release. They also correlate with weak, slow Ca2+ rhythms in non-AVP cells, such as VIP neurons.

We next built a simplified mathematical model consisting of two oscillators to test if the free-running period of normal mice (i.e., the synchronized, SCN ensemble period) could be explained by the periods of AVP- and VIP-Ca2+ rhythms measured in this study (Fig. 8b and Supplementary Fig. 11). Here, the VIP-Ca2+ period represented non-AVP cells. The oscillations were obtained using a phase oscillator model with coupling31. Based on the Ca2+ rhythms in VIP-deficient mice in DD (Fig. 2a), we estimated the in vivo intrinsic period lengths of the dominant fast oscillator (i.e., AVP neurons: τA) and the presumed weak slow oscillator (non-AVP cells: τnA) to be ~22.7 h and ~24.3 h, respectively. The free-running periods of Ca2+ and the locomotor activity rhythm in control mice were ~23.7 h (Figs. 1g and 2a) and regarded as the normal SCN ensemble period.

Since AVP neurons are dominant, the coupling strength from AVP to non-AVP cells (KA→nA) should be greater than the coupling strength from non-AVP to AVP cells (KnA→A). We set KA→nA = 5x KnA→A (α=5) based on an explant study13, or 25× KnA→A (α=25) considering the reports that AVP neurons may control other SCN cells more effectively in vivo than ex vivo16,17. As in the SNFL model, we paid particular attention to the phase-delaying effect of VIP (KV) by introducing a separate term to the AVP neuronal oscillator equation. We roughly estimated that VIP is released during subjective day (CT0~12) when VIP-Ca2+ is high (Fig. 1) (red underline in Fig. 8b). Additionally, based on the phase response curves of the SCN explant PER2::LUC rhythm to VIP application32 and the behavior rhythm to in vivo optogenetic stimulation of VIP neurons33, we assumed that AVP neurons respond to VIP around CT6–CT18 (green underline in Fig. 8b) primarily by delaying their phases. Using these equations, we identified parameter ranges that produce a synchronized ensemble period of ~23.7 h (Fig. 8b, bottom). In contrast, without the additional special term (Fig. 8b, KV = 0), two oscillators synchronized; however, the resultant period remained close to the short period of the AVP neuronal oscillator and never approached the measured value of ~23.7 h when the AVP cellular oscillator dominated the non-AVP cellular oscillator (α=5 or 25).

In VIP-deficient mice, VIP’s phase-delaying effect (KV) was reduced to zero, and both AVP (KA→nA) and non-AVP (KnA→A) cellular rhythms were weakened. Nevertheless, as far as the AVP neuronal oscillator dominated the non-AVP cellular oscillator (α=5 or 25; KnA→A is not too close to zero), the two oscillators synchronized, expressing a short period close to that of the AVP cellular oscillator. However, when the AVP cellular oscillator was attenuated to the point of losing control over non-AVP cells (α and KnA→A approaching zero), AVP and non-AVP cells oscillated with different short and long periods, respectively. This is similar to what was often observed in the later stage of DD. In Avp-Vipr2−/− mice, the synchronized period was shortened due to reduced KV. In contrast, in Avp-Bmal1−/− and Avp-TeNT mice, the periods lengthened due to reduced KA→nA (i.e., α=0 or 0.2). Furthermore, this model could similarly explain the period length of Avp-CK1δ−/−; Vipwt/tTA and Avp-CK1δ−/−; ViptTA/tTA mice by just lengthening the AVP cellular intrinsic period by 1–1.5 h (Supplementary Fig. 11d).

Thus, the SNFL model may appropriately represent the logic of SCN pacesetting, both qualitatively and quantitatively.

Methods
Animals
Avp-Cre (C57BL/6J-Tg(Avp-icre)#Meid/Rbrc, RBRC12048) and Vip-tTA (B6(Cg)-Vipem1(tTA2)Miem/Rbrc, RBRC12109) mice were reported previously10,12. Cck-ires-Cre (Ccktm1.1(cre)Zjh/J, JAX:012706)24, Rosa26-LSL-tdTomato (Ai14)(B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, JAX:007914)27, Rosa26-LSL-SpCas9-2A-EGFP (B6J.129(B6N)-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J, JAX:026175)29, Vip-ires-Cre(Viptm1(cre)Zjh/J, JAX:010908)24, CK1δ flox (B6.129S4-Csnk1dtm1Drw/J, JAX:010487)66, and Bmal1 flox (B6.129S4(Cg)-Bmal1tm1Weit/J, JAX:007668)67 mice were obtained from Jackson Laboratory. All lines were congenic on C57BL/6J. We compared the mutant mice with controls whose genetic backgrounds were comparable. Avp-Cre, Cck-ires-Cre, Vip-ires-Cre, Rosa26-LSL-tdTomato, and Rosa26-LSL-SpCas9-2A-EGFP mice were used in hemizygous or heterozygous conditions. We used both male and female mice in our experiments. Whether we pooled data from both sexes or analyzed them separately, the conclusions we reached remained almost the same. Previous studies reported slight but statistically significant sex differences in the behavior rhythms of VIP knockout mice (e.g., 0.05 h longer free-running period in females)68,69. However, the differences observed between genotypes in this study were large enough not to be affected by sex differences. The sex of individual mice is described in Supplementary Figs. and the Source Data file. Mice were maintained under a strict 12-h light/12-h dark cycle in a temperature- and humidity-controlled room and fed ad libitum. All experimental procedures involving animals were approved by the appropriate institutional animal care and use committees of Kanazawa University.

Viral vector and surgery
The AAV-2 ITR containing plasmid pGP-AAV-CAG-FLEX-jGCaMP7s-WPRE (Addgene plasmid #104495, a gift from Dr. Douglas Kim and GENIE Project)22, was obtained from Addgene. pAAV-TRE-ChR2-YFP (Addgene #171622, a gift from Hyungbae Kwon)70, was modified to construct pAAV-TRE-jRGECO1a by replacing ChR2-YFP cDNA with a jRGECO1a cDNA fragment amplified by PCR from the plasmid pAAV.CAG.-Flex.NES-jRGECO1a.WPRE.SV40 (Addgene #100852, a gift from Douglas Kim & GENIE Project)23, using the following primers: 5′-CTAGGATCCGCCACCATGCTGCAGAACGAGCTTGC-3’ and 5’-CTGAAGCTTTCACTTCGCTGTCATCATTTGTAC-3′. Then, an EcoRI-HindIII fragment of the resultant plasmid containing jRGECO1a sequence was used to replace an EcoRI-HindIII fragment containing ChrimsonR-mCherry from pAAV-TRE-ChrimsonR-mCherry (Addgene #92207, a gift from Alice Ting)71. pAAV-CAG-FLEX-taCasp3-TEVp was made by replacing a BamHI-EcoRV fragment containing EGFP of pAAV-CAG-FLEX-EGFP20 with a BamHI-EcoRV fragment containing taCasp3-TEVp from pAAV-flex-taCasp3-TEVp (Addgene #45580, a gift from Nirao Shah & Jim Wells)72. pAAV-U6-gVipr2-EF1α-DIO-mCherry, a plasmid for CRISPR-Cas9-mediated Vipr2 gene disruption, was generated as follows. The target sites for CRISPR-Cas9 were designed by CRISPOR73. Two sequences targeting Vipr2 gene were selected: 5′-TGCTGGCGCCCGGCAGACGT-3′ and 5′-CCAGATTTCATAGATGCGTG-3′. Oligonucleotides encoding the guide sequences were cloned into the BbsI and BsaI sites of pX333 (Addgene #64073, a gift from Dr. Andrea Ventura)74. Then, a fragment containing two tandem units of U6-gRNA was amplified by PCR, using the following primers: 5′-agtacgcgTCGAGCATGCTCGAGAATGG-3′ and 5′-agtacgcgtCGGGTACCCCATTTGTCTGC-3′, and cloned into the MluI site of pAAV-EF1a-DIO-mCherry (a gift from Dr. Bryan Roth) as described previously75. pAAV-U6-gControl-EF1α-DIO-mCherry contains spacer sequences from pX333 instead of gRNA sequences for Vipr2. pAAV-EF1α-DIO-GFP::TeNT was kindly provided by Dr. Richard D. Palmiter76.

Recombinant AAV vectors (AAV2-rh10) were produced using a triple-transfection, helper-free method and purified as described previously12. Briefly, 293A cells (Thermo Fisher Scientific) were transfected with pHelper (Stratagene), pAAV2-rh10 (provided by Penn Vector Core), and one of AAV-2 ITR containing plasmids using a standard calcium phosphate method. Three days later, the transfected cells were collected and suspended in PBS containing 1 mM MgCl2. After 2 freeze-thaw cycles, the cell lysate was treated with benzonase nuclease (Merck) at 37 °C for 30 min, and centrifuged 2 times at 16,000 × g for 10 min. The supernatant was used as the virus-containing solution. The titers of recombinant AAV vectors were determined by quantitative PCR: AAV-CAG-DIO-jGCaMP7s, 3.4×1013; AAV-TRE-jRGECO1a, 8.6×1012; AAV-CAG-FLEX-taCasp3-TEVp, 5.7×1012; AAV-CAG-FLEX-nls-mCherry, 6.1×1012; AAV-U6-gVipr2-EF1α-DIO-mCherry, 6.8×1012; AAV-U6-gControl-EF1α-DIO-mCherry, 2.6×1012; AAV-EF1α-DIO-GFP::TeNT, 1.8×1012 and AAV-CAG-FLEX-EGFP, 1.0×1013 genome copies/ml. Stereotaxic injection of AAV vectors was performed as described previously12. Two weeks or immediately after surgery, we began monitoring the mice for their locomotor activity.

In vivo fiber photometry
We used 8 Avp-Cre; ViptTA/tTA, 6 Cckwt/ires-Cre; ViptTA/tTA, 8 control (5 Avp-Cre; Vipwt/tTA and 3 Cckwt/ires-Cre; Vipwt/tTA), and 6 Avp-Bmal1−/−×Vip-tTA (Avp-Cre; Bmal1flox/flox; Vipwt/tTA) mice. Mice were anesthetized by administering a cocktail of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg) and were secured to the stereotaxic apparatus (Muromachi Kikai). Lidocaine (1%) was applied for local anesthesia before making the surgical incision. We drilled a small hole in the exposed region of the skull using a dental drill. We injected 1.0 μL of the mixture of AAV-CAG-FLEX-jGCaMP7s and AAV-TRE-jRGECO1a (ratio 1:1, flow rate=0.1 μL/min) into the right SCN (posterior: 0.5 mm, lateral: 0.25 mm, depth: 5.7 mm from the bregma) with a 33G Hamilton syringe (1701RN Neuros Syringe, Hamilton) to label AVP and VIP neurons. We then placed an implantable optical fiber (400 μm core, N.A. 0.39, 6 mm, ferrule 2.5 mm, Thorlabs or RWD) above the SCN (posterior: 0.2 mm, lateral: 0.2 mm, depth: 5.3 mm from the bregma) with dental cement (Super-bond C&B, Sun Medical). The dental cement was painted black. Atipamezole (0.3 mg/kg) was administered postoperatively to shorten the anesthetized period. Mice were used for experiments 2–7 weeks after the virus injection and optical fiber implantation. Their ages ranged from 3 to 10 months, including both males and females.

A fiber photometry system (FP3002, Neurophotometrics) was used to record the calcium signal of SCN neurons in freely moving mice77,78. Excitation light sources were a 470-nm LED for detecting Ca2+-dependent jGCaMP7s fluorescence signal (F470), a 560-nm LED for detecting Ca2+-dependent jRGECO1a fluorescence signal (F560), and a 415-nm LED for Ca2+-independent isosbestic fluorescence signal (F415). The duration of the excitation lights was 50 ms, and the onsets of the excitation timing of the LEDs were interleaved. The lights passed through excitation bandpass filters, dichroic mirrors, and then to the animal via fiber-optic patch cords (BBP(4)_400/440/900-0.37_1m_FCM-4xFCM_LAF, MFP_400/440/LWMJ-0.37_1m_FCM-ZF2.5_LAF, Doric Lenses) and the implanted optical fiber. Subsequently, all signals were detected using a CMOS camera through the optical fibers, dichroic mirrors, and emission bandpass filters. The green emission signal and the red emission signal were recorded separately and simultaneously. Ca2+ signals were recorded every 10 min for 30 s using Bonsai software at a sampling rate of 6.6 Hz for each color. The excitation intensities of the 470-nm, 560-nm, and 415-nm LEDs at the tip of the patch cord on the animal side ranged from 60 to 120 μw. During recording, the mouse was housed in a 12-h light–dark cycle for more than 7 days (LD condition) and then transferred to continuous darkness for more than 21 days (DD condition) in a custom-made acrylic cage surrounded by a sound-attenuating chamber.

Ratio (R) was defined as the ratio between F470 and F415 in green channel (F470/F415green) or the ratio between F560 and F415 in red channel (F560/F415red) for calibration and reducing motion artifacts. R was averaged within a 30-s session20. To detrend the gradual decrease of the signal during the recording days, ±12 h average from the time (145 points) was calculated as baseline (R0). The data were subsequently detrended by the subtraction of R0 (ΔR). Then, the ΔR/R0 value was calculated. To determine the daily peak phase of jGCaMP7s or jRGECO1a Ca2+ signal, ΔR/R0 were smoothened with a 21-point moving average, then the middle of the time points crossing value 0 upward (Ca2+ onset) and downward (Ca2+ offset) were defined as the midpoint that reflects the peak phases. Additionally, the intervals between two adjacent midpoints were defined as the daily periods (Fig. 1f, g)12. Because VIP-Ca2+ rhythm in VIP deficient mice attenuated drastically in DD, we failed to determine its Ca2+ onset, offset, and daily period at the late stage. A double-plotted actogram of the jGCaMP7s or jRGECO1a signal was prepared by converting all ΔR/R0 to positive values by subtracting the minimum value of ΔR/R0. Subsequently, these values were multiplied by 100 or 1000 and rounded off. The plots were made via ClockLab (Actimetrics) with normalization in each row to emphasize weak VIP-Ca2+ and CCK-Ca2+ rhythmicity in VIP-deficient mice at the late stage in DD. To quantitively analyze the weak rhythms and their changes, periods and amplitudes of Ca2+ signals (ΔR/R0) were calculated by periodogram with 10-min bins for all three weeks and shifting five-day time windows in DD, respectively (Figs. 2, 4, and 6; Supplementary Figs. 1, 2, 5, and 9).

During the fiber photometry recordings, the animal’s locomotor activity was monitored using an infrared sensor (AMN31112 and Arduino) in 10-min bins. A double-plotted actogram of locomotor activity was also prepared and overlaid on that of the jGCaMP7s and jRGECO1a signal. The onset of locomotor activity was determined using the actograms. Initially, we attempted to automatically detect the onset; however, it was followed by a manual visual inspection and modifications by the experimenter. The intervals between two adjacent locomotor onsets were defined as the daily periods (Fig. 1f, g). To calculate the CT of the midpoint phases of jGCaMP7s and jRGECO1a signals, we have defined the regression line of locomotor activity onsets as CT12. Period and amplitude of locomotor activity were also calculated by χ2 periodogram analyses for all days or 5 consecutive days in DD to analyze weak rhythmicity of VIP-deficient mice (Figs. 2, 4, and 6; Supplementary Figs. 1, 2, and 5).

We confirmed the jGCaMP7s and jRGECO1a expressions and the position of the optical fiber by slicing the brains into 30 μm coronal sections using a cryostat (Leica). The sections were mounted on glass slides with a mounting medium (VECTASHIELD HardSet with DAPI, H-1500, Vector Laboratories; Dako Fluorescence Mounting Medium, Agilent Technologies) and observed via epifluorescence microscope (BZ-9000, Keyence).

Behavioral analyses
Male and female Avp-CK1δ−/−; ViptTA/tTA (Avp-Cre; CK1δflox/flox; ViptTA/tTA) or control (Avp-Cre; CK1δflox/flox; Vipwt/tTA) (Fig. 2), Vip-Casp3 (Vipwt/ires-Cre mice injected bilaterally with AAV-CAG-FLEX-taCasp3-TEVp) or control (Vipwt/ires-Cre mice injected with AAV-CAG-FLEX-nls-mCherry and wild type C57BL/6J mice injected with AAV-CAG-FLEX-taCasp3-TEVp) (Supplementary Fig. 7), Avp-Vipr2−/− (Avp-Cre; Rosa26-LSL-Cas9-2A-EGFP mice injected bilaterally with AAV-U6-gVipr2-EF1α-DIO-mCherry) or control (Avp-Cre; Rosa26-LSL-Cas9-2A-EGFP mice injected with AAV-U6-gControl-EF1a-DIO-mCherry) (Fig. 5), and Avp-TeNT (Avp-Cre mice injected bilaterally with AAV-EF1α-DIO-GFP::TeNT) or control (Avp-Cre mice injected with AAV-CAG-FLEX-EGFP) (Fig. 7) mice, aged 8 to 30 weeks, were housed individually in a cage placed in a light-tight chamber (light intensity was approximately 100 lux). Spontaneous locomotor activity (home-cage activity) was monitored by infrared motion sensors (O’Hara) in 1-min bins with a custom-made program as described previously12. Actogram, activity profile, and χ2 periodogram analyses were performed via ClockLab (Actimetrics). The free-running period and amplitude were measured by periodogram for all days in DD (Avp-CK1δ−/−; ViptTA/tTA and Avp-TeNT) or the last 14 days in DD (Vip-Casp3). In the case of Avp-Vipr2−/− and control mice, their periods and amplitudes were measured by periodogram for 10 days in 1st or 2nd stage in DD (Fig. 5).

Immunohistochemistry
Immunostaining was performed as described previously10,12,75. Mice were killed approximately at ZT2 by transcardial perfusion of PBS followed by 4% paraformaldehyde (PFA) in PBS. For estimating co-expression of CCK and AVP, Cck-ires-Cre; Rosa26-LSL-tdTomato mice were pretreated with intracerebroventricular injections of colchicine (40 µg in 1 µl saline) 48 h before perfusion around ZT6. Then, the mice brains were postfixed in the 4% PFA at 4˚C overnight, followed by immersion in 30% sucrose solution at 4°C for 2 days. Serial coronal brain sections (30 μm thickness) were made with a cryostat (CM1860, Leica) and collected in 4 series—one of which was further immunostained. For immunofluorescence staining, sections were washed with PBS containing 0.3% Triton X-100 (PBST) and blocked with PBST plus 3% BSA (blocking solution). Then, slices were incubated overnight with the designated primary antibodies in the blocking solution at 4°C. Antibodies used were rabbit anti-GFP antibody (1:1000; A11122, Thermo Fisher Scientific), rabbit anti-VIP antibody (1:1,000; #20077, Immunostar), rabbit anti-AVP antibody (Millipore, 1:2000), and rabbit anti-VPAC2 antibody (Abcam, 1:1000). Then, slices were washed with PBST, followed by incubation with the designated secondary antibodies in blocking solution for 4 h. Secondary antibodies used in this study were Alexa Fluor 488–conjugated donkey anti-rabbit IgG antibody (1:2000, A-21206; Thermo Fisher Scientific), Alexa Fluor 594–conjugated donkey anti-rabbit IgG antibody (1:2000, A-21207; Thermo Fisher Scientific), and Alexa Fluor 647–conjugated goat anti-Rabbit IgG antibody (1:600, 111-605-003; Jackson Immuno Research Laboratories). After incubation with secondary antibody, slices were washed with PBS, mounted on slide glasses, air dried, and coverslipped using Mounting Medium (H-1500, Vector Laboratories; Dako Fluorescence Mounting Medium, Agilent Technologies). Images were taken using an epifluorescence microscope (BZ-9000, Keyence) or a confocal microscope (Fluoview Fv10i, Olympus). Fluorescent signal of Alexa Fluor 647 was pseudo-colored in light blue (Fig. 5 and Supplementary Fig. 7). The VPAC2 expression levels were quantified by ImageJ as follows (Fig. 5b). First, the mean intensities of pixels within the ROIs in the SCN shell and core were calculated. Then, the values of the region lateral to the SCN were regarded as background and were subtracted from those of the SCN.

Mathematical model construction and simulation
The two-oscillator model with coupling was modified by introducing a term in which VIP neurons delay the phase of AVP neuron oscillator31, reflecting the phase-dependent receptivity of AVP to VIP’s delaying effect.

dθA/dt = ωA + KnA→A sin(θnA − θA) − KV * max(sin(θnA), 0) * max(sin(θA − δ), 0)

dθnA/dt = ωnA + KA→nA sin(θA − θnA)

where θA, θnA denote the phases of oscillator A (representing AVP neurons) and oscillator nA (non-AVP neurons, including VIP neurons), respectively. ωA, ωnA are the intrinsic angular velocities (ωA = 2π/τA, ωnA = 2π/τnA), and τA, τnA are the intrinsic periods (τA = 22.7 h, except for Supplementary Fig. 11d where τA = 23.7 h; τnA = 24.3 h), based on the AVP-Ca2+ and VIP-Ca2+ fiber photometry results presented in this study. KnA→A denotes the coupling strength from oscillator nA to A, KA→nA denotes the coupling strength from oscillator A to nA, and KV represents inhibitory modulation (e.g., VIP-mediated inhibition), which is active only when sin(θnA) > 0 and sin(θA − δ) > 0. δ represents the phase difference between oscillator A and its receptivity, and was set to π/2 (Fig. 8b), 0 (Supplementary Fig. 11a), π (Supplementary Fig. 11b), or 3π/2 (Supplementary Fig. 11c). The ratio between KA→nA and KnA→A (α) was set to 0, 0.2, 1, 5, and 25. In the heatmaps, gray regions indicate simulations in which a stable period could not be determined, whereas black regions indicate the final periods between oscillators A and nA are different (>0.2 h). All simulations were performed in MATLAB.

Statistical analysis
All results are expressed as mean±SEM. Statistical analyses were performed using Prism 8.0 software (GraphPad). For comparisons of 2 groups, two-tailed unpaired Student’s or Welch’s t tests were performed. For comparisons of multiple groups with no difference of variance by Bartlett test, two-way repeated measures ANOVA followed by Bonferroni, Tukey or Holm-Sidak post hoc test were performed. For circular data, Rayleigh test, Watson–Williams test, and Harrison–Kanji test were performed with Circstat MATLAB Toolbox for Circular Statistics79. All p values less than 0.05 were considered as statistically significant. Only relevant information from the statistical analysis was indicated in the text and figures. Detailed results of the statistical analyses are provided in Supplementary Data 1.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "How the 20,000 neurons and astrocytes of the suprachiasmatic nucleus (SCN) generate a stable ~24 hours (circadian) signal is an important area of study because this property determines the temporal pattern of our behavior and physiology. In this study, Wang et al. use an impressive range of intersectional manipulations and long-term in vivo Ca-imaging to explore how two distinct cell groups of the SCN, those expressing arginine vasopressin (AVP) and those expressing vasoactive intestinal peptide (VIP) interact to main robust rhythmicity. They conclude that “the neuronal feedback circuit composed of AVP cellular oscillator and VIP peptide signaling is crucial for the robust circadian rhythm.”\nI have two significant thoughts about his study. First, the impressive technical features of in vivo cell-specific imaging have already been published by this group, including the excellent paper by Tsuno et al, PLOS Biology, 2023. This reduces some of the immediate impact of the study. Second, the conclusion could have been made before this work was conducted. There is an extensive literature, again some of it from this group, about the role of AVP neurons in driving circadian behavior and of the requirement for VIP signaling to sustain the SCN clockwork. The real question to be answered is how do AVP and VIP cells do what they do - what are the mechanisms? It is not clear to me how this study moves towards a substantial answer, but the Authors may be able to articulate how their results do push things forward by elucidating “the mechanism of how VIP regulates the cellular clocks of AVP neurons”.\n1. Figures 1 & 2 show how Ca2+ rhythms in SCN VIP and AVP cells relate to each other and to behavioral rhythms in wild-type mice, and then how these relationships possibly change in the VIP-deficient ViptTA mouse. Specifically, it shows that loss of VIP reduces the amplitude and shortens the period of AVP Ca rhythms and, in parallel, is associated with disrupted behavioral activity rhythms with overall shortened period.\nEven under LD the Ca rhythms of AVP cells are diminished in VIP-null mice. This is an interesting result because it indicates that, in the absence of VIP, light cannot boost the AVP Ca rhythms. Thus, loss of normal VIP signaling affects the SCN circuit properties and the period of AVP cells.\nIn the absence of VIP, Ca rhythms in VIP cells remain robustly light-driven but disappear very rapidly on transfer to DD. Why might VIP cells be sensitive to loss of VIP because scRNAseq has shown that they do not express Vipr2? The Authors suggest this is an indirect result of the weaker and faster AVP cell rhythms in VIP-null mice, hence the common period of 23.67 hours for AVP and VIP cells.\nIn the final stages of the study, however, VIP cells are reported to show a longer period of 24.33 hours in VIP-null mice. I do not fully understand, however, how the VIP Ca rhythms were analysed. In Figure 1d, e, it petered out after 1 week of DD but in Figure 2 parameters are calculated until days 16 -20. Is it not possible that these parameter values are meaningless because the rhythms have become so poorly defined? A value may be calculated for period, but it will come with an error term that may render it not biologically meaningful. Similarly, the periodograms in Figure 2a suggest a dual periodicity (early and later?) for VIP Ca rhythms – but how these were calculated and analysed statistically is not clear. The time-series plots in Figure 2c show the evolution of the longer period, but the point remains that these rhythms were very poorly defined and so how much relevance can be placed on this apparent change in period? In addition, in the moving time series, the same data points are used repeatedly, and then ANOVA is applied to the means for each 5 days value. I am unclear as to whether this is appropriate as the measures are not independent, for example 5-9 shares 4 values of 6-10.\nA final query is why does the later behavioral period track the presumed late period of the VIP and not the period of the AVP cells (Figure 2c) if AVP cells are the pacesetters? Over the 20 days of recording the amplitude and period of AVP cells are unchanged and so it might be expected that their influence over behavior would also be maintained.\nIn the later stages of recording are we simply looking at some non-specific associations in a clock that is progressively falling apart: behavioral offsets could not even be defined in the VIP-null mice? In the early stages of DD, where measurements are much more robust, AVP and VIP cells behave similarly.\nI have a technical question regarding the individual period values. Is there some aspect of the periodogram which causes it to deliver particular fractional values? For example, Figure 2a has .67 (2/3) and .33 (1/3), as does Figure 3e, alongside .50 (1/2). To what degree does the doublet for period in VIP-null animals apply to individual animals and to what degree is it a population measure?\n“These results suggest that VIP neurons may be a subordinate slow oscillator and exhibit a weak, long-period rhythm that correlates with the behavior rhythm in vivo when the AVP cellular rhythm is attenuated by VIP deficiency.”\nAlternatively, it may be that the loss of VIP in some way alters the intrinsic properties of VIP cells. Moreover, the Authors do not consider whether changes in the Ca rhythms are a result of altered behavioral rhythms and not a cause of them.\n2. To test whether the VIP-null compromise of AVP (shorter period) and VIP cell (short then long period) rhythms is generalisable across the SCN, Figure 3 describes Ca2+ rhythms in SCN CCK cells and the impact of VIP deletion on them. Again, elegant intersectional approaches are deployed. In control mice, the CCK cells exhibit a Ca rhythm, as reported previously. The VIP-null phenotype of VIP cells was replicated with an independent cohort of mice. Note, is this assumed independence correct, even though the mice again had identical periods of 22.67 h and 24.33 h (see technical comment above re. periodogram)? In VIP-null mice the rhythms are initially light-driven and then damp rapidly on transfer to DD. They do lengthen, but not to the extent seen in Figure 1, raising uncertainly about the robustness of the late VIP-null phenotype.\nThe Ca rhythms of CCK cells in VIP-null mice were light-driven on LD and showed rapid damping in DD, replicating the phenotype of VIP cells. They do not, however, show a late lengthened period, as the VIP cells did, so they are a half-way house. The statistical comparisons are pretty weak, however, and again suffer from the poor definition/ high variance of rhythms in the later stages. The Authors conclude that “CCK-Ca2+ rhythm is weak and unstable and has more heterogeneous periods in VIP deficiency. Such heterogeneity may partly reflect that CCK neurons contain at least two subpopulations, one overlapping with a small subpopulation of AVP neurons.” This is speculation. Fundamentally in the absence of VIP the SCN cellular rhythms are low amplitude (AVP) or damped poorly defined period (VIP, CCK).\n3. Given the importance of VIP when absent throughout development, the Authors then explore the effect of adult-restricted loss of the VIP cells. A different mouse line, heterozygous VIP IRES Cre received AAV injection of Cre-dependent caspase. Post-mortem staining revealed absence of VIP, and the mice exhibited a short period of 22.9 hours. Unfortunately, there are no control animals in this experiment: for example, injected with control AAV or mice not of the IRES-CRE background and injected with AAV conditional caspase. The data are therefore incomplete, and this particular study would require expansion to be suitable for publication.\n4. To complement the examination of VIP-deficiency, the Authors then deleted its receptor, Vipr2, from AVP cells by cell-specific expression of Cre-conditional CAS9 and AAV inject of sgRNA. (Figure 4) This is, again, a technically impressive approach although targeting was confirmed by mCherry and GFP signals but levels of Vipr2 were not determined. This targeting caused an acute shortening of period, compared to mice injected with control AAV, but given the impact of genomic ablation of Vipr2 (short period and/ or highly disturbed activity rhythms and a desynchronised SCN) it is surprising that the phenotype is so modest. Despite the short period, the activity patterns are robust, and behavioral period is recovered after ~ 3weeks. This was repeated in a second cohort of ice – something changes ~21 days after injection. In the absence of validation of the deletion of Vipr2, it is difficult to interpret these data.\nThe Authors state that: “These observations suggest the indirect pathways in the regulation of AVP neurons by VIP neurons that may be involved in the compensatory recovery of the standard period in Avp-Vipr2−/− mice.” This is highly speculative.\n5. Having targetted Vipr2 in AVP cells, the Authors then tested the effect of Bmal1 deletion in AVP cells on behavior and VIP and AVP cell Ca rhythms. On my reading, there is no control group in this experiment: all mice were AVP-Bmal1 null? This confounds any interpretation of the phenotypes observed: period, duration of Ca elevation etc.\nNotably, the targetted animals exhibited a longer period, 24.17 h, but this is far off the original report of 24.8 +0.1 hours in Mieda et al 2015. And again, a single period is given but with no error terms to mirror its variance between mice.\nThe Authors note that: “As expected, the AVP-Ca2+ exhibited little circadian rhythmicity, although this might be due to low jGCaMP7s expression in TTFL-deficient AVP neurons.” So, is the rhythm correctly reported or not? It is very clear that robust VIP cell Ca rhythms are maintained, again with an identical period to behavior, 24.17 hours. So ostensibly, when AVP cells have no TTFL, VIP cells can nevertheless drive circadian behavior?\n6. In the final element of the study, the Authors blocked neurotransmitter release from AVP cells by injecting Cre-dependent AAV TeNT, or AAV EGFP as a control. This caused a clear and unequivocal lengthening of period (~24.5 hours) when compared to controls (~23.2 hours). This is again interpreted as the effect of releasing the SCN from its dominance by the intrinsically short-period oscillator of AVP cells. There are also interesting aspects of the relative movements of “evening” and “morning” activity bouts, and the phenotype chimes very well, as noted by the Authors, with AVP-Bmal1 null and AVP-Vgat null.\n7. “Our results reveal a feedback loop neuronal circuit of these two neuron types that may be the essential network mechanism of the SCN central circadian pacemaker.”\nThe conclusion is an extension of the literature, but this proposal fails to indicate mechanisms. The nature of the loop is not specified: what is its structure and topology, and how might it sit with regard to other SCN circuit components? There is a sense of playing “whack a mole” insofar as when reasoning from VIP cells to AVP cells or vice versa, additional unknown and indirect elements have to be hypothesised to fill the gaps such that the identity to the relevant circuit elements is never pinned down.\n8. On a general point to facilitate presentation, I suggest that the figures and text should be rearranged in order to report on control conditions first (placed to the left and at the top of figures) and then say how experimental groups (placed to the right and at the bottom) compared with controls.", "reviewer_2": "The study applied dual-color fiber photometry recording to VIP-deficient mice, allowing them to simultaneously monitor the cellular circadian rhythms from two types of SCN neurons. They analyzed the circadian behavior rhythms of multiple mouse strains that had various impairments in the communication between VIP and AVP neurons.\nThis is an extremely elegant and complex genetic approach, to disrupt VIP (and other) signaling while performing dual-color Ca2+ fiber photometry. The technological approach is very impressive. The results are complex. One caveat that should be, and is, addressed in the text is that the assumption that the populations of VIP and AVP (and CCK) cells are internally coherent, especially during disruption of normal signaling, may not be true. For example see https://www.pnas.org/doi/10.1073/pnas.2209329120. Plus, recent snRNAseq studies have shown that VIP+ and AVP+ populations are internally diverse. Therefore, treating them as distinct groups may be an oversimplification. I don’t suggest that it invalidates these results, just that it should be clearly acknowledged that there is likely more complexity/diversity among cells and across days. The beginning of the discussion does acknowledge this, which is good, as it is a key point.\nThe results are, generally, wide in terms of # of manipulations, but shallow, in terms of #s of mice or depth of understanding of the system, derived from a disrupting VIP signaling, blocking the TTFL in cell subtypes, etc. Studying the coupled system in vivo, while it is broken, is tough to do before we understand how it works when it isn’t broken. The discussion reveals that the paper raises way more questions than it can answer. Overall this is a technical tour de force, with rather limited new insight achieved. No new conceptual model is provided that can help us understand this circuit better than we already did.", "reviewer_3": "Summary of the key results\nThe researchers investigated the role AVP neurons play in SCN regulation. They hypothesize that VIP -> AVP communication plays a role in circadian rhythm, given that the TTFL loop in VIP neurons is weaker compared to that in AVP neurons. The research shows that AVP neurons are the principal oscillators with an intrinsically short period. It strengthens previous observations by this research group. When the internal clock of AVP neurons is disrupted by Bmal1 KO or their communication with other neurons is removed with TeNT, the rhythm becomes longer than 24 hours. These results shed interesting new light on the role of AVP neurons.\nI think the interpretation of some unique features that appeared in the results could be claimed in a better way if additional data will be considered and presented, as listed below.\nOriginality and significance:\nThe paper, as indicated in the discussion, is the first to use fiberphotometry with 2-colors in the SCN in vivo. The paper nicely combines genetic manipulation and AAV-based technologies (such as CRISPR) with calcium imaging and LMA (locomotor activity) detection. Furthermore, the paper’s significance lies in the in vivo monitoring of AVP-related features. The fact that in vivo effects are different from slices is critical here, as the SCN and AVP neurons also get inputs from the surrounding, which was not tested or discussed here, which is a weakness of the paper.\nThe paper aims to understand the communication mechanism, but it lacks a clear model, or at least it is not represented in a clear way. To specify, the fact that AVP is a short-period pacemaker is well explained and demonstrated, but the interpretation of the changes in the LMA split or VIP calcium imaging after 3 weeks is not strongly convincing. An additional figure that shows the model will be a great addition.\nData & methodology:\n1. The combined usage of calcium imaging, LMA detection, and cell manipulation using AAV is impressive. The data analysis is straightforward, but could benefit from a more advanced analysis, which will highlight the dual activity along the day, such as Rayleigh plots, as presented in Supp. Figure 2.\n2. In the paper by Wen et. al. (https://www.nature.com/articles/s41593-020-0586-x) it was shown that Cck neurons are divided into two subpopulations, one is rhythmic Cck+/C1ql3+ and one is less (Cck+/Bdnf+). Did you try to identify which subpopulation was located under the fiber? See, for example, https://www.cell.com/cell-reports/fulltext/S2211-1247(21)01197-9, or even regular slice histology. This could shed light on the effect found in vivo.\n3. The low number of animals in Figure 5 (n=3) makes it hard to interpret the results.\n4. The results under DD conditions in the manipulated animals are interpreted as a phase-delaying model, and the two oscillators option is declined by considering a few aspects. However, I suggest that the new LMA pattern suggests split SCN activity (Figure 4). It was found in hamsters https://pubmed.ncbi.nlm.nih.gov/11760012/, and was identified as a split SCN activity by https://www.science.org/doi/full/10.1126/science.290.5492.799.\nI would be interested in seeing circadian clock gene expression during different time points throughout the day. Is the expression even on both sides of the SCN under DD?\n5. When genetic cell manipulation was used using AAV (Figure 4, Figure 6), did you check if the results depend on the virus injection spread pattern? Was one side of the SCN better expressed by the virus compared to the other? The quality of the injection (with reference to Right/Left) vs. the effect should be quantified, and the interpretation should include this information.\n6. It is not clear to me why the initial hypothesis is that the prevention of the neurotransmitters' release (Figure 6) would lengthen the period. I would expect it will prevent cell-cell communication and will cause asynchrony, which will show unsynchronized LMAs at DD, when there is no entrainment. I would further claim that the diversity in the results could be due to Right/Left expression of the virus. The histology of AAV efficiency in each mouse should be determined and presented in correlation to the effect.\nConclusions:\n1. At the full animal organism, other aspects should be taken into account. For example, what was the effect on melatonin? What was the core body temperature? To emphasize this point, in Figure 1, the LMA in VIP-deficient #1 seems to overlap with VIP-Ca+2 signal after day ~20, which is very unique. It is also reflected in Figure 2c. Is it possible that VIP neurons are more sensitive to entrainment, and when light entrainment is not present, activity (maybe temperature) will entrain the TTFL cycle?\n2. Some critical papers are missing from the Discussion, such as a previous paper showing VIP-/- activity and comparison to it (https://journals.sagepub.com/doi/10.1177/0748730414549767) and AVP single-cell imaging in vivo https://www.pnas.org/doi/epub/10.1073/pnas.2209329120\nMinor:\n1. In the abstract: “VIP and other neurons occasionally exhibit weak, unstable, long-period calcium rhythms only when AVP neuronal oscillation is attenuated. ” – only when- not clear. Compared to what?\n2. In page 5: “In VIP-deficient mice, the AVP-Ca2+ rhythm was almost correlated with the short-period behavior rhythm in DD” – this is not obvious from Figure 1. For example, Figure 1d shows VIP Ca+2 offset correlates with locomotor onset. Figure 1e shows a significant difference between AVP-Ca+2 and locomotor activity. Please clarify.\n3. As for the disappearing rhythm of Ca+2 signal of VIP neurons in VIPtTA/tTA mice– could it be due to VIP neurons' apoptosis? As VIP is deficient in these mice, I would suggest that the neurons had some decline in their health, especially when combined with GCaMP, which is toxic. Did you check VIP neuronal health after imaging?\n4. It is not clear how Figure 1C and 1F include VIP Ca signal for 25 days, but the analysis in d and e is only up to day 14.\n5. Why Figure 1 and 2 are separated? They both show the same experiment with continuous analysis approaches.\n6. Based on Figure 1 and Supp Figure 1, some mice had a shift towards ~24h rhythm after day ~18, and some did not. Did you try to analyze the cohort separated into two groups, based on this observation? Do you think it can be predicted based on a feature in their LD activity?\n7. Figure 6: E and M should be indicated in Figure 6c.\n8. In the Discussion: each of populations - > each one of the populations / each population\n9. In the Discussion: “Nevertheless, this method is so far the only one that allows long-term recordings of SCN dynamics in genetically defined neuronal populations in vivo.” – this sentence is not correct; see, for example, https://www.pnas.org/doi/epub/10.1073/pnas.2209329120\n10. Pg. 13- SNFL - not defined\n11. While the paper did not mention differences between males and females, this should be considered, given the differences found here: https://www.biorxiv.org/content/10.1101/2022.08.18.504454v2.abstract"}, "author_response": "Response to Reviewers\nThe authors would like to thank all reviewers for their valuable comments and suggestions,\nwhich guided us in significantly improving our manuscript. In the revised manuscript, we\nadded a section to the Results that describes the SNFL model, along with model figures\n(Fig. 8), explaining the importance of this study in proposing the model. Furthermore, we\nbuilt a simplified mathematical model that simulates the pacesetting by the SNFL.\nAnother significant addition in the revised manuscript is the direct evidence of a causal\nrelationship between the AVP neuronal clock and the short-period behavior rhythm in\nVIP-deficient mice (Fig. 3).\nOur detailed responses to the reviewers’ concerns are itemized below.\nResponse to Reviewer #1\n1-1. How the 20,000 neurons and astrocytes of the suprachiasmatic nucleus (SCN)\ngenerate a stable ~24 hours (circadian) signal is an important area of study because this\nproperty determines the temporal pattern of our behavior and physiology. In this study,\nWang et al. use an impressive range of intersectional manipulations and long-term in vivo\nCa-imaging to explore how two distinct cell groups of the SCN, those expressing arginine\nvasopressin (AVP) and those expressing vasoactive intestinal peptide (VIP) interact to\nmain robust rhythmicity. They conclude that “the neuronal feedback circuit composed of\nAVP cellular oscillator and VIP peptide signaling is crucial for the robust circadian\nrhythm.”\nI have two significant thoughts about his study. First, the impressive technical features of\nin vivo cell-specific imaging have already been published by this group, including the\nexcellent paper by Tsuno et al, PLOS Biology, 2023. This reduces some of the immediate\nimpact of the study. Second, the conclusion could have been made before this work was\nconducted. There is an extensive literature, again some of it from this group, about the\nrole of AVP neurons in driving circadian behavior and of the requirement for VIP\nsignaling to sustain the SCN clockwork. The real question to be answered is how do AVP\nand VIP cells do what they do - what are the mechanisms? It is not clear to me how this\nstudy moves towards a substantial answer, but the Authors may be able to articulate how\ntheir results do push things forward by elucidating “the mechanism of how VIP regulates\nthe cellular clocks of AVP neurons”.\nResponse: The authors appreciate the reviewer’s crucial comments. We are glad that the\nreviewer does not reject our SNFL model, but rather considers it reasonable and capable\nof explaining the previously reported results in an extensive literature. While we, as\nwell as Prof. Joe Takahashi’s group, have proposed the dominant role of AVP neurons as\nthe circadian oscillator in vivo and ex vivo, respectively (Mieda et al., Neuron 2015, PMID:\n25741730; Mieda et al., Curr Biol 2016, PMID: 27568590; Tsuno et al., Plos Biol 2023,\nPMID: 37643163; Shan et al., Neurons 2020, PMID: 32768389), we do not feel that this\nview is widely accepted in the field (e.g., Hastings et al., Nat Rev Neurosci 2018; Patton\net al., Nat Commun 2020; Nikhil et al., bioRxiv 2024, PMID: 39713450). Due to the\nimpactful role of VIP peptide as a critical synchronizer of SCN cells, many researchers\nlikely still consider VIP neurons as the dominant circadian oscillator in the SCN network,\na notion that has yet to be demonstrated in vivo. We believe that our SNFL model, built\nupon previous works and novel findings in this study, bridges the gap between these two\nlines of research. Therefore, as explained below, it provides significant conceptual\nprogress in understanding the neural mechanism of the mammalian central circadian\nclock.\nWe added a section “Neuronal feedback loop model of the SCN explains experimental\nresult” and a figure (Fig. 8a) to the Results to explain the SNFL model and clarify its\nnovel points. We also included a simplified mathematical model that simulates the control\nof period length by the SNFL (Fig. 8b). Our model combines the new findings from this\nstudy with two lines of previously reported results. First, AVP neurons serve as the\nprimary pacesetter. The other result is that VIP signaling is essential, though the\ncontribution of VIP neurons as oscillators is minimal. Qualitatively, VIP release,\ncontrolled by AVP neuronal rhythm, daily amplifies and delays the intrinsically short-\nperiod AVP neuronal rhythm, bringing it close to a 24 h-period, under the free-running\ncondition in DD (i.e., the endogenous SCN rhythm in the absence of any external\nzeitgeber, such as a light pulse). Thus, the model centers on a single, primary, short-period\noscillator (AVP neuron), and feedback signals (VIP signaling), driven by this oscillator,\nprolong the endogenous period of the oscillator, thereby modulating the overall SCN\nperiod to 24 hours.\nThe key novel findings of this study to complete our SNFL model are as follows:\n#1: AVP neurons maintain an intrinsically short-period, low-amplitude rhythm in vivo\neven in the absence of VIP signaling.\n#2: AVP neurons are responsible for the short-period behavior rhythm in VIP-deficient\nmice.\n#3: VPAC in AVP neurons contributes significantly to the phase delay of the AVP\n2\nneuronal rhythm.\nIf it were only such a phase-delay/amplification feedback loop, it would be\nstraightforward. However, we also obtained the following result:\n#4: Attenuated, long-period behavior and VIP-Ca2+ rhythms remain when the AVP\nneuronal oscillator is attenuated by VIP deficiency, or AVP neuron-specific disruption of\nTTFL or transmitter release.\nThis #4 suggests that non-AVP cells, including VIP neurons, also function as oscillators,\nalbeit marginally in the presence of SNFL, which complicates the interpretation. Here,\nsuch weak oscillations are incorporated into the unified SNFL model by considering them\nas the default clock state (a term inspired by the default mode network) or background\ncircadian oscillation (a term inspired by cosmic background radiation). These oscillations\nbecome visible in the absence of SNFL. In mammals, almost every cell throughout the\nbody has a TTFL, as do all SCN cells. Thus, it is not surprising that weak circadian\nrhythms emerge when the primary oscillator (AVP neurons) loses control over the rest of\nthe SCN cellular oscillators. We incorporate all SNFL components in a mathematical\nmodel by adding a term expressing the phase-delaying effect of VIP to a Kuramoto model\nequation of two-oscillator coupling: one strong AVP neuronal oscillator with a short\nperiod and one weak non-AVP cellular oscillator (including VIP neurons) with a long\nperiod (Fig. 8b).\nWithout this extra term (K = 0), the model alone cannot explain the experimental results\nV\nof this study. Suppose the ensemble period is set according to a simple coupling of\nstronger fast and weaker slow oscillators. In that case, the ensemble period would be\nmuch closer to that of the fast oscillator, which does not fit with the experimental results\n(i.e., AVP-Ca2+, ~22.7 h; VIP-Ca2+, ~24.3 h, as the representative of non-AVP cells;\nensemble, ~23.7 h, the Ca2+ and behavioral periods in normal mice). Adding the extra\nterm replicates the recorded period lengths very well. We would like to emphasize that\nthe model is based on the values measured by long-term, simultaneous, in vivo recordings\nof neuron-type-specific Ca2+ rhythms (i.e., AVP, VIP, or CCK neurons) and behavioral\nrhythms, which has been done for the first time in this study. This scheme is quite flexible\nin setting the ensemble period by adjusting the phase-delaying power of VIP signaling,\neven longer than the long-period weak oscillator (“Besides, the SNFL scheme is highly\nflexible in setting the ensemble period by adjusting the phase-delaying power of VIP\nsignaling (K ), ...” in the Discussion). Therefore, it may be suitable for light entrainment\nV\nby transiently modifying the VIP signaling intensity and subsequently the ensemble\nperiod to cause phase shifts in both directions.\nIn our previous report (Tsuno et al., Plos Biol 2023, PMID: 37643163), we proposed that\nthe cellular circadian period of AVP neurons is the primary determinant (i.e., pacesetter)\nof the SCN ensemble period in vivo. This view remains unchanged in this study, albeit\nwith an update reflecting that the ensemble period length is ~1 h longer than the intrinsic\nAVP cellular period due to the phase-delaying effect of VIP signaling (“This view\nremained unchanged in this study but was further updated to account for the ensemble\nperiod length...” in the Discussion). We were able to make this update because we\nsuccessfully estimated the in vivo intrinsic AVP neuronal period using dual-color fiber\nphotometry recordings in VIP-deficient mice. In this sense, AVP neurons are not fully\nautonomous pacesetters. Rather, they are equipped with feedback neural circuits that\nperfect their role as the pacesetter. This idea provides a significant conceptual\nadvancement.\nIndeed, as the reviewer pointed out, there are past reports that suggest a part of the\nelements that make up our model. However, as far as we know, no similar model has ever\nbeen proposed. In this study, we identified the final critical pieces that complete our model.\nWe agree that previous studies have demonstrated the necessity of VIP signaling for\nsustaining the SCN clockwork, which served as the starting point of our current study.\nVIP application or VIP neuronal activation delays PER2::LUC rhythms in explants (An\net al., J Neurophysiol 2011, PMID: 21389307; Patton et al., Nat Commun 2020, PMID:\n32636383) and behavior rhythms in vivo (Mazuski et al., Neuron 2018, PMID: 30017392;\nXie et al., Neuron 2023, PMID: 37172583). However, these effects have primarily been\ndiscussed in the context of light entrainment (“which has been regarded as the\ncellular/molecular mechanism of phase-delay by the external light pulse” in the\nDiscussion). Besides, based on the extensive projection of VIP neurons to the SCN shell\nand the high (but not exclusive) expression of VPAC in the shell, researchers postulate\n2\nthat VIP resets the SCN ensemble phase by acting on the SCN shell. Nevertheless, there\nis no direct evidence of this, nor of the identity of the direct target cells that mediate the\nphase reset among the many VPAC -expressing cells. Some studies have described the\n2\nreciprocal interaction between the SCN core and shell in the SCN explants, again\nprimarily in the context of responses to the phase-shifting light pulses, the extreme\nphotoperiods, and the coupling of shell and core oscillators of comparable strength (Albus\net al., Curr Biol 2005, PMID: 15916945; Evans et al., Neuron 2013, PMID: 24267653;\nMyung et al., PNAS 2015, PMID: 26130804).\nIt should be noted that this model does not exclude the functions of AVP neurons and VIP\nneurons as the output arms of the SCN, such as those regulating timed drinking behavior\n(Gizowski et al., Nature 2016, PMID: 27680940), neurosecretion via the SCN-OVLT\nportal system (Yao et al., Nat Commun 2021, PMID: 34561434), and corticosterone\nrelease (Paul et al., Nat Commun 2020, PMID: 32193397; Jones et al., Nat Commun 2021,\nPMID: 34599158). This situation may be analogous to the intracellular timekeeping,\nwherein the TTFL ticks the cellular clock and also drives output clock-controlled genes\nin individual cells (“However, this model does not exclude the involvement of extra-SCN\nneurons, because the SCN also receives inputs from its surroundings” in the Discussion).\nWe made a maximum effort to clarify the novelty of the current study in the revised\nmanuscript. However, the reviewer’s instructions and suggestions would be more than\nwelcome to further improve its readability.\nRegarding the reviewer’s comment on the technical features of this study, we feel that it\nwas undervalued. The other two reviewers agreed that our strategy for this study is\ntechnically novel and excellent. As mentioned earlier, in vivo multicolor measurements\ncombined with locomotor activity monitoring for over three weeks allowed us to directly\ncorrelate the SCN dynamics and behavior rhythms for the first time, which cannot be\ndone with SCN explants. Besides, we examined multiple neuron type-specific genetic\nmanipulations, including AVP neuron-specific Vipr2 knockout, which has not been done\npreviously.\nAs the reviewer pointed out, the precise molecular mechanisms of how VIP regulates the\ncellular clocks of AVP neurons is the critical question. Given the importance of VPAC\n2\nin AVP neurons, as demonstrated here, we speculate on the involvement of the cAMP\ncascade and Per induction, as has been suggested for light entrainment and VIP effects\non the SCN. Some expected molecular mechanisms, based on the previous reports, were\ndiscussed (“The exact intracellular events downstream of VPAC in AVP neurons to delay\n2\ntheir TTFLs remain elusive...” in the Discussion). What exactly is happening in AVP\nneurons remains to be determined. However, this will require in vivo analyses, which are\ntechnically more challenging than those using an ex vivo system. Therefore, we would\nlike to pursue this important topic as the next step in our research.\n1-2. Figures 1 & 2 show how Ca2+ rhythms in SCN VIP and AVP cells relate to each other\nand to behavioral rhythms in wild-type mice, and then how these relationships possibly\nchange in the VIP-deficient ViptTA mouse. Specifically, it shows that loss of VIP reduces\nthe amplitude and shortens the period of AVP Ca rhythms and, in parallel, is associated\nwith disrupted behavioral activity rhythms with overall shortened period.\nResponse: In DD, the ability of AVP neurons as the pacesetter is likely around the\nthreshold due to their attenuated rhythm in the absence of amplification by VIP signaling.\nAbove the threshold, AVP neurons retain some control over other SCN cells and the\nbehavior rhythm, thereby pacesetting a short-period rhythm. Below the threshold,\nhowever, AVP neurons lose control over other SCN cells, allowing weak, long-period\nrhythms dissociated from AVP neuronal rhythms. We expanded the discussion on this\ntopic and also clarified it in the model figures and the mathematical model (Fig. 8).\nThe heterogeneity of the circadian phenotype in VIP-deficient mice is well known, but the\ncause is unclear. Thus, our model may propose an explanation for it.\n1-3. Even under LD the Ca rhythms of AVP cells are diminished in VIP-null mice. This is\nan interesting result because it indicates that, in the absence of VIP, light cannot boost\nthe AVP Ca rhythms. Thus, loss of normal VIP signaling affects the SCN circuit properties\nand the period of AVP cells.\nResponse: We appreciate the valuable comment. We agree with this point and added the\nsentence: “In LD, light may not appropriately entrain and boost the AVP-Ca2+ rhythm in\nthe absence of VIP.” (in the Results).\n1-4. In the absence of VIP, Ca rhythms in VIP cells remain robustly light-driven but\ndisappear very rapidly on transfer to DD. Why might VIP cells be sensitive to loss of VIP\nbecause scRNAseq has shown that they do not express Vipr2? The Authors suggest this is\nan indirect result of the weaker and faster AVP cell rhythms in VIP-null mice, hence the\ncommon period of 23.67 hours for AVP and VIP cells.\nResponse: We agree with this point and added the sentence “Such a feedback loop circuit\nmay also explain the observation that the VIP-Ca2+ rhythm quickly attenuated in DD in\nthe absence of VIP even though VIP neurons express little VPAC .” (in the Discussion).\n2\n1-5. In the final stages of the study, however, VIP cells are reported to show a longer\nperiod of 24.33 hours in VIP-null mice. I do not fully understand, however, how the VIP\nCa rhythms were analysed. In Figure 1d, e, it petered out after 1 week of DD but in Figure\n2 parameters are calculated until days 16 -20. Is it not possible that these parameter\nvalues are meaningless because the rhythms have become so poorly defined? A value may\nbe calculated for period, but it will come with an error term that may render it not\nbiologically meaningful. Similarly, the periodograms in Figure 2a suggest a dual\nperiodicity (early and later?) for VIP Ca rhythms – but how these were calculated and\nanalysed statistically is not clear. The time-series plots in Figure 2c show the evolution\nof the longer period, but the point remains that these rhythms were very poorly defined\nand so how much relevance can be placed on this apparent change in period? In addition,\nin the moving time series, the same data points are used repeatedly, and then ANOVA is\napplied to the means for each 5 days value. I am unclear as to whether this is appropriate\nas the measures are not independent, for example 5-9 shares 4 values of 6-10.\nResponse: In Fig. 1, we characterized the behavior rhythms by actograms and the Ca2+\nrhythms by the curve lines of detrended, smoothened jGCaMP7s/jRGECO1a signals, as\ndescribed in the Methods. As the reviewer pointed out, we gave up calculating the values\nfor VIP-Ca2+ after a week in DD because it became too weak and noisy to accurately\nanalyze by this method. At the same time, we observed the signs of weak rhythms at the\nlate stage in actograms. Therefore, in Fig. 2, we used the periodogram analysis to extract\nsuch weak rhythms objectively and statistically. We further clarified the procedure in the\nmanuscript (Results, Methods, and Legends). We agree with the reviewer that such weak\nrhythms may not be biologically meaningful. We merely demonstrated the presence of\n“statistically significant” rhythms. To clarify this point, we added the following sentences\nto the Results and Discussion: “However, the causal relation between these weak VIP-\nCa2+ and behavior rhythms remains unknown,” “their weak, slow Ca2+ rhythm may be a\npassive, merely correlated oscillation.” In fact, it was from this point that we came up\nwith the idea of the background circadian oscillation. In contrast, we confirmed the causal\nrelationship between the AVP neuronal rhythm and the short-period behavior rhythm in\nVIP-deficient mice in the revised manuscript (Fig. 3).\n1-6. In addition, in the moving time series, the same data points are used repeatedly, and\nthen ANOVA is applied to the means for each 5 days value. I am unclear as to whether\nthis is appropriate as the measures are not independent, for example 5-9 shares 4 values\nof 6-10.\nResponse: We believed that overlapping shifting windows would express the time course\nof period changes more precisely. The purpose of these analyses is not to show significant\noscillation at a certain point, but rather, to compare the time course of period changes\nbetween genotypes. Therefore, we did not consider this procedure statistically\ninappropriate. Nevertheless, to avoid such a concern, we also analyzed with multiple time\nwindows without overlap and reached the same conclusion (Supplementary Fig. 1f,g). If\nthe reviewer prefers this presentation, we would be happy to replace the graphs.\n1-7. A final query is why does the later behavioral period track the presumed late period\nof the VIP and not the period of the AVP cells (Figure 2c) if AVP cells are the pacesetters?\nOver the 20 days of recording the amplitude and period of AVP cells are unchanged and\nso it might be expected that their influence over behavior would also be maintained.\nResponse: As previously mentioned (1-2), the ability of AVP neurons as to act as the\npacesetter is likely around the borderline in VIP-deficient mice. It is not surprising that\nthe amplitude of the AVP-Ca2+ rhythm gradually reduces in prolonged DD, although it is\nmaintained. Such a tendency is seen in Fig. 1d and Fig. 2e. It is well known that VIP- or\nVPAC2-deficient mice show phenotypic heterogeneity, single short-period or multiple-\nperiod circadian rhythm. The reviewer’s question may be similar to one we are often\nasked by researchers: Why do VIP-deficient mice exhibit two types of behavioral\nrhythms? The slight difference in AVP neurons’ pacesetting ability may cause such a\nspectrum of circadian phenotypes in these mice.\n1-8. In the later stages of recording are we simply looking at some non-specific\nassociations in a clock that is progressively falling apart: behavioral offsets could not\neven be defined in the VIP-null mice? In the early stages of DD, where measurements are\nmuch more robust, AVP and VIP cells behave similarly.\nResponse: As described above (1-5), we agree with the reviewer that the weak, late-onset\nVIP-Ca2+ rhythms have no biological meaning. Although AVP- and VIP-Ca2+ rhythms\nbehave similarly in the early stages, they are attenuated as compared to normal mice,\nwhich may lead to the attenuation of the behavior rhythm as well, and thereby make it\ndifficult to define behavioral offsets in VIP-deficient mice. To ensure consistency, the\nrevised Fig. 1 excludes locomotor offset data from the control mice as well.\n1-9. I have a technical question regarding the individual period values. Is there some\naspect of the periodogram which causes it to deliver particular fractional values?\nFor example, Figure 2a has .67 (2/3) and .33 (1/3), as does Figure 3e, alongside .50 (1/2). To\nwhat degree does the doublet for period in VIP-null animals apply to individual animals\nand to what degree is it a population measure?\nResponse: Because the jGCAMP7s/jRGECO1a signal was recorded every 10 minutes,\nthe Ca2+ rhythm data are in 10-min bins, i.e., 1/6 h. Therefore, we performed periodogram\nanalyses with 10-min bins. In Fig. 2a, we first averaged the periodograms of individual\nmice and identified the primary and secondary peaks of period lengths. For this reason,\nthe value is a multiple of 1/6 without standard errors. Since the degree of double\nperiodicity varied from mouse to mouse, we averaged all periodograms to objectively\nextract the period lengths. To confirm this result, we also performed the analysis in reverse\norder in the revised manuscript. That is, we first calculated the periods for each mouse\nand then averaged them (Fig. 2b,c). The detailed values were different, of course, but the\nconclusions were the same.\n1-10. “These results suggest that VIP neurons may be a subordinate slow oscillator and\nexhibit a weak, long-period rhythm that correlates with the behavior rhythm in vivo when\nthe AVP cellular rhythm is attenuated by VIP deficiency.”\nAlternatively, it may be that the loss of VIP in some way alters the intrinsic properties of\nVIP cells. Moreover, the Authors do not consider whether changes in the Ca rhythms are\na result of altered behavioral rhythms and not a cause of them.\nResponse: The authors appreciate this critical comment. As described above (1-5), we\ndo not aim to claim that the weak oscillation of VIP neurons drives the weak long-period\nbehavior rhythm.\n1-11. 2. To test whether the VIP-null compromise of AVP (shorter period) and VIP cell\n(short then long period) rhythms is generalisable across the SCN, Figure 3 describes Ca2+\nrhythms in SCN CCK cells and the impact of VIP deletion on them. Again, elegant\nintersectional approaches are deployed. In control mice, the CCK cells exhibit a Ca\nrhythm, as reported previously. The VIP-null phenotype of VIP cells was replicated with\nan independent cohort of mice. Note, is this assumed independence correct, even though\nthe mice again had identical periods of 22.67 h and 24.33 h (see technical comment above\nre. periodogram)? In VIP-null mice the rhythms are initially light-driven and then damp\nrapidly on transfer to DD. They do lengthen, but not to the extent seen in Figure 1, raising\nuncertainly about the robustness of the late VIP-null phenotype.\nResponse: The reason that the value is a multiple of 1/6 is the same as explained above\n(1-9). We measured different cohorts of VIP-deficient mice between Fig. 1/2 and Fig. 3,\nand we think the VIP-null phenotype is consistent. The reviewer may be criticizing the\napparent difference between Fig. 2d and 4g. As the reviewer pointed out, the late VIP-\nnull rhythmicity varies between individuals, resulting in big error bars. We repeatedly\nexpressed this rhythm as “weak” and never claimed it was robust. We added a sentence\n“Although 5 days may be too short to precisely calculate the period length, it may be\nsufficient to briefly track the time course of changing periods” in the Results.\n1-12. The Ca rhythms of CCK cells in VIP-null mice were light-driven on LD and showed\nrapid damping in DD, replicating the phenotype of VIP cells. They do not, however, show\na late lengthened period, as the VIP cells did, so they are a half-way house. The statistical\ncomparisons are pretty weak, however, and again suffer from the poor definition/ high\nvariance of rhythms in the later stages. The Authors conclude that “CCK-Ca2+ rhythm is\nweak and unstable and has more heterogeneous periods in VIP deficiency. Such\nheterogeneity may partly reflect that CCK neurons contain at least two subpopulations,\none overlapping with a small subpopulation of AVP neurons.” This is speculation.\nFundamentally in the absence of VIP the SCN cellular rhythms are low amplitude (AVP)\nor damped poorly defined period (VIP, CCK).\nResponse: We completely agree with the reviewer that “Fundamentally in the absence of\nVIP the SCN cellular rhythms are low amplitude (AVP) or damped poorly defined period\n(VIP, CCK).” In the revised manuscript, we demonstrated that the low amplitude AVP\ncellular rhythm is responsible for the short-period behavior rhythm in VIP deficient mice\n(Fig. 3). As for VIP and CCK cellular rhythms, we clarified that we are showing just\ncorrelations, as explained above (1-5). Concerning subpopulations of CCK neurons, we\nadded an experiment estimating the overlap of AVP and CCK neurons in the revised\nmanuscript (Supplementary Fig. 6).\n1-13. 3. Given the importance of VIP when absent throughout development, the Authors\nthen explore the effect of adult-restricted loss of the VIP cells. A different mouse line,\nheterozygous VIP IRES Cre received AAV injection of Cre-dependent caspase.\nPost-mortem staining revealed absence of VIP, and the mice exhibited a short period of 22.9\nhours. Unfortunately, there are no control animals in this experiment: for example,\ninjected with control AAV or mice not of the IRES-CRE background and injected with\nAAV conditional caspase. The data are therefore incomplete, and this particular study\nwould require expansion to be suitable for publication.\nResponse: We appreciate the reviewer’s comment. We added appropriate control mice in\nthe revised manuscript (Supplementary Fig. 7).\n1-14. 4. To complement the examination of VIP-deficiency, the Authors then deleted its\nreceptor, Vipr2, from AVP cells by cell-specific expression of Cre-conditional CAS9 and\nAAV inject of sgRNA. (Figure 4) This is, again, a technically impressive approach\nalthough targeting was confirmed by mCherry and GFP signals but levels of Vipr2 were\nnot determined. This targeting caused an acute shortening of period, compared to mice\ninjected with control AAV, but given the impact of genomic ablation of Vipr2 (short period\nand/ or highly disturbed activity rhythms and a desynchronised SCN) it is surprising that\nthe phenotype is so modest. Despite the short period, the activity patterns are robust, and\nbehavioral period is recovered after ~ 3weeks. This was repeated in a second cohort of\nice – something changes ~21 days after injection. In the absence of validation of the\ndeletion of Vipr2, it is difficult to interpret these data.\nThe Authors state that: “These observations suggest the indirect pathways in the\nregulation of AVP neurons by VIP neurons that may be involved in the compensatory\nrecovery of the standard period in Avp-Vipr2−/− mice.” This is highly speculative.\nResponse: We confirmed a substantial, but incomplete, reduction of VPAC in the SCN\n2\nshell of Avp-Vipr2−/− mice by immunostaining (Fig. 5b,c). The phenotype of these mice\nwas indeed much less severe than reports of conventional Vipr2 KO mice. We also\nobserved a less severe phenotype in VIP neuron-ablated mice during adulthood as\ncompared to conventional Vip KO mice (Supplementary Fig. 7), consistent with a\nprevious report by Mazuski et al. (J Biol Rhythms 2020, PMID: 32536240), which\nimplicates some developmental effects of VIP/VPAC signaling. Besides, Vipr2 was\n2\ndeleted only in SCN AVP neurons, which had not been done before. We modified the\nsentences in the revised manuscript as “These observations may be due to an incomplete\nelimination of VPAC from AVP neurons. In addition, we speculate that the indirect\n2\npathways may exist that mediate the regulation of AVP neurons by VIP neurons and are\ninvolved in the compensatory recovery of the standard period in Avp-Vipr2−/− mice” in\nthe Results.\n1-15. 5. Having targetted Vipr2 in AVP cells, the Authors then tested the effect of Bmal1\ndeletion in AVP cells on behavior and VIP and AVP cell Ca rhythms. On my reading, there\nis no control group in this experiment: all mice were AVP-Bmal1 null? This confounds\nany interpretation of the phenotypes observed: period, duration of Ca elevation etc.\nResponse: We consider the similar recordings of control mice in Fig. 1 and 2 (Avp-Cre;\nBmal1wt/wt; Vipwt/tTA) to be the control also for Avp-Bmal1−/− mice in Fig. 6 (Avp-Cre;\nBmal1flox/flox; Vipwt/tTA). Because repeated usage of the same data should be avoided, we\ndid not present the control data in the original manuscript. Nevertheless, in the revised\nmanuscript, we presented a representative actogram (Fig. 6c) different from Fig. 1c (but\nincluded in the analysis of Fig. 1/2) and the mean values of ΔR/R values (Fig. 6d,e) of\n0\ncontrol mice for comparison. Concerning the periodogram (Fig. 6f,g), the same recorded\ndata were analyzed differently because of a reason explained in the following reply (1-\n16). We clearly declared the reuse of data in the text and legend. In addition, we increased\nthe number of Avp-Bmal1−/− mice recorded in the revised manuscript.\n1-16. Notably, the targetted animals exhibited a longer period, 24.17 h, but this is far off\nthe original report of 24.8 +0.1 hours in Mieda et al 2015. And again, a single period is\ngiven but with no error terms to mirror its variance between mice.\nResponse: The apparent difference of periods may be derived from the instability of free-\nrunning rhythms in Avp-Bmal1−/− mice (Mieda et al., Neuron 2015, PMID: 25741730).\nTo run periodogram analyses, we used data for the last 14 days (the second and third\nweeks) in DD. However, as reported in Mieda et al., Avp-Bmal1−/− mice take ~2 weeks\nbefore their activity time reaches a steady-state length. Until then, the onset of the\nbehavior rhythm free-runs with a period shorter than that of behavior offset. In addition,\nthe free-running period of Avp-Bmal1−/− mice often fluctuates and further lengthens\nunder prolonged DD, as reported previously. The data might contain those before the\nsteady state. To avoid this confounding factor, we reanalyzed with the last 7 days data in\nDD. Besides, We recorded additional Avp-Bmal1−/− mice in the revised manuscript.\nThe resultant free-running period was 24.33 h (Fig. 6f). The reason for a single period without\nerror terms is as explained earlier (1-9). Additionally, 10-min bins used for periodogram\nanalyses slightly affected the resultant peak period length. Because we recorded the Ca2+\nsignals every 10 min, we also analyzed the behavior rhythm with 10-min bins. When 1-\nmin bins are used for the behavior rhythm, the resultant free-running period is 24.43 h.\nThese are the values obtained by averaging periodograms, as in Fig. 2a. Even with 10-\nmin bins, another calculation presented in Fig. 5g, in which the peak period lengths were\nfirst calculated for individual mice and then averaged as in Fig. 2b, gave us the value\n24.47 ± 0.16 h. This value is still smaller than 24.8 h in a precise sense, but they are almost\ncomparable. The slight difference due to the calculation method may also indicate the\ninstability of the period in Avp-Bmal1−/− mice and reflect the weakness of the\nbackground circadian oscillation described in the SNFL model. Indeed, these three values\nfor control mice are 23.67 h, 23.68 h, and 23.60 ± 0.09 h. In any case, the qualitative\nnature of our SNFL model would not change. Here, control mice data were also\nreanalyzed for the last 1 week in DD, which might partially avoid the repeated\npresentation of the same data.\n1-17. The Authors note that: “As expected, the AVP-Ca2+ exhibited little circadian\nrhythmicity, although this might be due to low jGCaMP7s expression in TTFL-deficient\nAVP neurons.” So, is the rhythm correctly reported or not? It is very clear that robust\nVIP cell Ca rhythms are maintained, again with an identical period to behavior, 24.17 hours.\nSo ostensibly, when AVP cells have no TTFL, VIP cells can nevertheless drive circadian\nbehavior?\nResponse: We apologize for the confusing phrase. We confirmed the expression of\njGCaMP7s and removed that phrase. Yes, even when AVP neurons have no TTFL, weak,\nslower oscillators of non-AVP neurons, including VIP neurons, remain, which can drive\nweak circadian behavior with a longer period. We thoroughly discussed and proposed the\nmodel in Fig. 8.\n1-18. 6. In the final element of the study, the Authors blocked neurotransmitter release\nfrom AVP cells by injecting Cre-dependent AAV TeNT, or AAV EGFP as a control. This\ncaused a clear and unequivocal lengthening of period (~24.5 hours) when compared to\ncontrols (~23.2 hours). This is again interpreted as the effect of releasing the SCN from\nits dominance by the intrinsically short-period oscillator of AVP cells. There are also\ninteresting aspects of the relative movements of “evening” and “morning” activity bouts,\nand the phenotype chimes very well, as noted by the Authors, with AVP-Bmal1 null and\nAVP-Vgat null.\nResponse: We appreciate the reviewer’s positive comment. We expanded the description\nand discussion in the revised manuscript regarding the altered phase relation between E\nand M locomotor activities in the Results and Discussion.\n1-19. 7. “Our results reveal a feedback loop neuronal circuit of these two neuron types\nthat may be the essential network mechanism of the SCN central circadian pacemaker.”\nThe conclusion is an extension of the literature, but this proposal fails to indicate\nmechanisms. The nature of the loop is not specified: what is its structure and topology,\nand how might it sit with regard to other SCN circuit components? There is a sense of\nplaying “whack a mole” insofar as when reasoning from VIP cells to AVP cells or vice\nversa, additional unknown and indirect elements have to be hypothesised to fill the gaps\nsuch that the identity to the relevant circuit elements is never pinned down.\nResponse: The SCN is a very complex structure, and no one has revealed an entire view\nof its clear circuit structure. While this study does not provide a complete picture of the\nSCN circuit mechanism, asking for that from one paper seems excessive. As mentioned\nabove (1-1), we believe that we have made important new findings in this study and have\nbeen able to integrate the previously reported results into a new model. We are unaware\nof any previous papers that explicitly propose the feedback loop circuit of the SCN for\ngenerating the free-running rhythm.\n1-20. 8. On a general point to facilitate presentation, I suggest that the figures and text\nshould be rearranged in order to report on control conditions first (placed to the left and\nat the top of figures) and then say how experimental groups (placed to the right and at\nthe bottom) compared with controls.\nResponse: We rearranged the order to report the results as suggested in the revised\nmanuscript.\nResponse to Reviewer #2\n2-1. The study applied dual-color fiber photometry recording to VIP-deficient mice,\nallowing them to simultaneously monitor the cellular circadian rhythms from two types\nof SCN neurons. They analyzed the circadian behavior rhythms of multiple mouse strains\nthat had various impairments in the communication between VIP and AVP neurons.\nThis is an extremely elegant and complex genetic approach, to disrupt VIP (and other)\nsignaling while performing dual-color Ca2+ fiber photometry. The technological\napproach is very impressive. The results are complex. One caveat that should be, and is,\naddressed in the text is that the assumption that the populations of VIP and AVP (and\nCCK) cells are internally coherent, especially during disruption of normal signaling,\nmay not be true. For example see https://www.pnas.org/doi/10.1073/pnas.2209329120. Plus,\nrecent snRNAseq studies have shown that VIP+ and AVP+ populations are internally\ndiverse. Therefore, treating them as distinct groups may be an oversimplification. I don’t\nsuggest that it invalidates these results, just that it should be clearly acknowledged that\nthere is likely more complexity/diversity among cells and across days. The beginning of\nthe discussion does acknowledge this, which is good, as it is a key point.\nResponse: We appreciate the reviewer’s positive comment. Regarding the diversity of\nAVP and VIP neurons, we expanded the discussion in the revised manuscript (the first\nparagraph of the “Discussion” section). We acknowledge that recent snRNA-seq studies\nhave significantly advanced our understanding of SCN neuron heterogeneity, identifying\nnumerous subpopulations distinguishable by their gene expression profiles. However,\ndeveloping genetic tools to specifically manipulate individual subpopulations\nnecessitates considerable additional effort. Even though such simplification is a limitation\nof this study, we believe this research is at the cutting edge of what is currently possible.\nRegarding the internal coherence of VIP and AVP neuronal populations, we\n(AVP neurons and VIP neurons) and others (VIP neurons) successfully observed\nsignificant daily rhythms of Ca2+ by in vivo fiber photometry. Therefore, it is rational to\nconclude that these cells are internally coherent (synchronized) to some extent at the\ncircadian frequency, despite having certain diversity in phase and amplitude of circadian\noscillation. This should be true even during the disruption of normal signaling, though the\ncoherence may be further attenuated. Does the reviewer think it’s possible that, in a\ncoupled system, the population has a rhythm while the individual components have no\nrhythm?\nStowie et al. (https://www.pnas.org/doi/10.1073/pnas.2209329120) is an\nexcellent, elegant, and impressive study, achieving technically challenging single-cell\nrecordings of Ca2+ rhythms of SCN AVP neurons in free-moving mice. Their conclusion\nthat most individual AVP neurons do not exhibit circadian baseline Ca2+ rhythms in both\nLD and DD somewhat contradicts to our conclusion (“assumption” by the reviewer) of\nsome circadian coherence between AVP neurons. Even at the population level (mean\nfluorescence values for all ROIs), they concluded that AVP neurons do not show a\nsignificant circadian rhythm of intracellular Ca2+ level in DD. On the other hand, and\nimportantly, they also described that the majority of bootstrap trials, which were\nperformed to address the unequal contribution of cells from each mouse, suggested a\npopulation-level circadian rhythm in both LD and DD. This bootstrap result might suggest\nthat there was a significant difference in the quality of single-cell recordings between\nindividual mice used. Thus, their data do not necessarily deny the population-level\ncircadian rhythm in AVP neurons.\nEven regarding the single cell-level rhythm, what we failed to grasp in their\npaper is as follows. Fig. 1A reported that only 6 out of 26 cells demonstrated significant\nrhythmicity in DD in the intensity of GCaMP signals. On the other hand, Supplemental\nTable S1 lists all cells recorded. Among 50 cells recorded in DD, 23 cells showed\nsignificant rhythmicity in the intensity of GCaMP signals. Intriguingly, 15 cells recorded\nin a mouse included 4 rhythmic cells, 11 cells in the second mouse included 2 rhythmic\ncells, and 24 cells in the third mouse included 17 rhythmic cells. Thus, we were not sure\nwhich of the 23 cells in Fig. 1A corresponded to which cells in Table S1. Moreover, we\nalso wondered about the substantial differences among the three recorded mice in the\nnumber of recorded cells and the proportion of rhythmic cells.\nBesides, Xie et al. (Neuron 2023, PMID: 37172583) reported the in vivo\ncircadian rhythm of Fos expression, which often reflects the intracellular Ca2+ level, in\nSCN AVP neurons. Although Enoki et al. (PNAS 2017, PMID: 28270612) reported robust\ncircadian Ca2+ rhythms in individual AVP neurons of SCN explants, results by Stowie et\nal. might indicate that in vivo conditions drastically destabilize and attenuate Ca2+\nrhythms in each AVP neuron. Nevertheless, it is hard to imagine that there is rhythm at\nthe population level but no rhythm at all at the individual cell level. At the same time, the\nrhythm in individual cells could be buried in noise due to the low signal-to-noise ratio\ncaused by the technical difficulty of the single-cell recording. While we recognize the\nlimitation of our current study, as described above and in the first paragraph of the\nDiscussion in the manuscript, there might be some confounding factors in Stowie et al.,\nincluding the small number of cells recorded, low expression level of GCaMP7s (the\nstrain Avp-ires2-Cre used has been known for low level expression of Cre and reduction\nof endogenous Avp expression: Cheng et al., J Biol Rhythms 2019, PMID: 31452438),\nlower sampling frequency (every 3-h recording), and the short-term (2 days) recording.\nWe discussed the apparent discrepancy between their and our studies in the second\nparagraph of the Discussion.\n2-2. The results are, generally, wide in terms of # of manipulations, but shallow, in terms\nof #s of mice or depth of understanding of the system, derived from a disrupting VIP\nsignaling, blocking the TTFL in cell subtypes, etc. Studying the coupled system in vivo,\nwhile it is broken, is tough to do before we understand how it works when it isn’t broken.\nThe discussion reveals that the paper raises way more questions than it can answer.\nOverall this is a technical tour de force, with rather limited new insight achieved. No new\nconceptual model is provided that can help us understand this circuit better than we\nalready did.\nResponse: We provided model figures and further built a simplified mathematical model\nto clarify the new idea derived from this study (Fig. 8). We described in detail the new\ninsight and conceptual advance made by the current study in our response to the first\ncomment by Reviewer #1. Because it is very long, we will not repeat it here, but ask the\nreviewer to look at the communication as well (1-1).\nWe agree with the reviewer’s opinion, “Studying the coupled system in vivo,\nwhile it is broken, is tough to do before we understand how it works when it isn’t broken.”\nOn the other hand, comparing the broken system with the intact system is often very\npowerful to understand the complicated intact system, such as studying knockout mice to\nunderstand the function of a gene of interest. Otherwise, observing only the intact systems\ntends to be just descriptive, without elucidating the causal relationships. Indeed,\nrecordings in VIP-deficient mice successfully dissolved the stable, fast AVP neuronal\noscillator and weak, slow non-AVP cellular oscillators, owing to the elimination of the\nlinkage between multiple oscillators. Our new techniques provided key findings essential\nfor proposing the SNFL model, which has not been presented before. The reviewers'\ncomments make critical points, but they also seem overly demanding for a single paper.\nThis research has certainly raised many new questions, and we hope to gain a deeper\nunderstanding of the mechanisms of the SCN central clock by seeking answers to these\nquestions in the future.\nRegarding the number of mice analyzed, we increased the number of mice\nrecorded in Fig. 6 and Supplementary Fig. 7. Because long-term in vivo fiber photometry\nrecordings are technically much more difficult than simple behavior rhythm recordings,\nwe think that the number of mice used in this study is acceptable (e.g., n=3 for some\nexperiments in Xie et al., Neuron 2023, PMID: 37172583).\nResponse to Reviewer #3\n3-1. Summary of the key results\nThe researchers investigated the role AVP neurons play in SCN regulation. They\nhypothesize that VIP -> AVP communication plays a role in circadian rhythm, given that\nthe TTFL loop in VIP neurons is weaker compared to that in AVP neurons. The research\nshows that AVP neurons are the principal oscillators with an intrinsically short period. It\nstrengthens previous observations by this research group. When the internal clock of AVP\nneurons is disrupted by Bmal1 KO or their communication with other neurons is removed\nwith TeNT, the rhythm becomes longer than 24 hours. These results shed interesting new\nlight on the role of AVP neurons.\nI think the interpretation of some unique features that appeared in the results could be\nclaimed in a better way if additional data will be considered and presented, as listed\nbelow.\nResponse: We appreciate the reviewer’s positive comments.\n3-2. Originality and significance:\nThe paper, as indicated in the discussion, is the first to use fiberphotometry with 2-colors\nin the SCN in vivo. The paper nicely combines genetic manipulation and AAV-based\ntechnologies (such as CRISPR) with calcium imaging and LMA (locomotor activity)\ndetection. Furthermore, the paper’s significance lies in the in vivo monitoring of AVP-\nrelated features.\nThe fact that in vivo effects are different from slices is critical here, as the SCN and AVP\nneurons also get inputs from the surrounding, which was not tested or discussed here,\nwhich is a weakness of the paper.\nResponse: We appreciate the reviewer’s valuable comment. We added a discussion about\nthis point in the revised manuscript: “Because AVP and VIP neurons are reciprocally\nconnected directly and indirectly, the intra-SCN network may be sufficient for SNFL\nfunction. However, this model does not exclude the involvement of extra-SCN neurons,\nbecause the SCN also receives inputs from its surroundings.”\n3-3. The paper aims to understand the communication mechanism, but it lacks a clear\nmodel, or at least it is not represented in a clear way. To specify, the fact that AVP is a\nshort-period pacemaker is well explained and demonstrated, but the interpretation of the\nchanges in the LMA split or VIP calcium imaging after 3 weeks is not strongly convincing.\nAn additional figure that shows the model will be a great addition.\nResponse: We added model figures and a brief mathematical model to explain the results\nand our idea better (Fig. 8). Regarding LMA (locomotor activity) split, we previously\ndemonstrated that the lack of GABA release from AVP neurons, due to specific Vgat\ndeletion, causes LMA split (expansion of the activity time), without changing the free-\nrunning period (Maejima et al., PNAS 2021, PMID: 33526663). In these mice, the TTFL\nand AVP-Ca2+ rhythms in the SCN appeared normal, but the phase relationships between\nAVP-Ca2+ and circadian morning (M) / evening (E) locomotor activities were\nsignificantly altered. Thus, GABAergic transmission of AVP neurons contributes to the\nphase-setting of LMA. Because both the pacesetting and phase-setting functions of AVP\nneurons were impaired in Avp-Bmal1−/− and Avp-TeNT mice, the LMA rhythm\ndemonstrated both long period and split. The observation that the split was more severe\nin Avp-TeNT than Avp-Vgat−/− may indicate the involvement of transmitters other than\nGABA in the phase-setting. At the same time, the existence of both E and M locomotor\nactivities in many Avp-TeNT mice, albeit split, suggests that AVP neurons are unlikely to\ncontain neurons (or E and M oscillators) that send the timed output signals to the\nlocomotion center. Rather, AVP neurons may transmit the timing information to output\nneurons, indicating when locomotor activity should occur. We also agree with the\nreviewer’s comment on the late-onset VIP-Ca2+ rhythm and LMA. As described in the\nresponse to Reviewer #1 (1-5), we do not aim to claim the causal relation between these\ntwo. We expanded these discussions in the revised manuscript (the paragraph starting with\n“TeNT expression in AVP neurons not only lengthened the free-running period but also\naltered the phase relationship of E and M locomotor activities….” in the Discussion).\nWe recognize that how the SCN regulates E and M LMA is a fundamental question in the\nfield. We would like to pursue this issue further, starting with the phase-setting function\nof AVP neurons. It should be noted that the phase-setting mechanism of AVP neurons is\nstill compatible with the encoding of photoperiod by the phase distribution of SCN clock\nneurons (the second-to-last sentence in the same paragraph).\n3-4. Data & methodology:\n1. The combined usage of calcium imaging, LMA detection, and cell manipulation using\nAAV is impressive. The data analysis is straightforward, but could benefit from a more\nadvanced analysis, which will highlight the dual activity along the day, such as Rayleigh\nplots, as presented in Supp. Figure 2.\nResponse: We may be misinterpreting the reviewer’s point. We could show Rayleigh\nplots in Supplementary Fig. 1 for the initial days in DD when the Ca2+ and behavior\nrhythms in VIP -deficient mice were relatively coherent. However, it was difficult to run\nthe same analyses at the late stage in DD because VIP-Ca2+ rhythms were so weak and\nnoisy.\n3-5. 2. In the paper by Wen et. al. (https://www.nature.com/articles/s41593-020-0586-x)\nit was shown that Cck neurons are divided into two subpopulations, one is rhythmic\nCck+/C1ql3+ and one is less (Cck+/Bdnf+). Did you try to identify which subpopulation\nwas located under the fiber? See, for example, https://www.cell.com/cell-\nreports/fulltext/S2211-1247(21)01197-9, or even regular slice histology. This could shed\nlight on the effect found in vivo.\nResponse: We thank the reviewer for raising an important point. The method developed\nby Kahan et al. (Cell Rep 2021, PMID: 34592157) presents an elegant strategy for\nlocalizing the optic fibers and GRIN lenses in single-cell Ca2+ imaging, where the depth\nof focus planes is shallow. In our fiber photometry, in contrast, the optic fibers were\nintended to be positioned just dorsal to the SCN to capture light from the wide area of the\nSCN. Therefore, it seems challenging to discriminate between two subpopulations of\nCCK neurons solely according to the fiber position. Practically, the CCK-Ca2+ signal was\nlikely derived from the mixture of these two. Instead, we added data about how much\nAVP and CCK neurons overlap in the SCN in the revised manuscript (Supplementary Fig.\n6). We conceived this analysis from the data by Wen et al. (Nat Neurosci 2020, PMID:\n32066983), indicating a low level Avp expression in the Cck+/C1ql3+ subpopulation.\nThese data would help clarify the molecular characteristics of CCK neuronal populations\nrecorded in this study.\n3-6. 3. The low number of animals in Figure 5 (n=3) makes it hard to interpret the results.\nResponse: We increased the number of mice recorded to 5. Because long-term in vivo\nfiber photometry recordings are technically more difficult than simple behavior rhythm\nrecordings, we think that the numbers of mice used in the revised manuscript are\nacceptable (e.g., n=3 for some experiments in Xie et al., Neuron 2023, PMID: 37172583).\n3-7. 4. The results under DD conditions in the manipulated animals are interpreted as a\nphase-delaying model, and the two oscillators option is declined by considering a few\naspects. However, I suggest that the new LMA pattern suggests split SCN activity\n(Figure 4). It was found in hamsters https://pubmed.ncbi.nlm.nih.gov/11760012/, and\nwas identified as a split SCN activity by\nhttps://www.science.org/doi/full/10.1126/science.290.5492.799.\nI would be interested in seeing circadian clock gene expression during different time\npoints throughout the day. Is the expression even on both sides of the SCN under DD?\nResponse: We think that the reviewer mentioned the LMA in Fig. 7. We briefly tested the\nreviewer’s idea by Per1 in situ hybridization. Although it was not thorough, we found no\nsign of left-right asymmetric Per1 expression in the SCN, as shown in the image below.\nBesides, we have already examined the clock gene expression in the SCN of Avp-Vgat−/\n− mice, which demonstrate clear splitting, by Per1 ISH and PER2::LUC SCN explant\nimaging. We observed their normal circadian expression, without any right/left\nasymmetry (Maejima et al., PNAS 2021, PMID: 33526663). Similarly, in Avp-Bmal1−/−\nmice, which also exhibit splitting in addition to the period lengthening, Per1, Prok2, and\nRgs16 mRNA expression, as well as explant PER2::LUC rhythm, in the SCN did not\nshow right/left asymmetry (Mieda et al., Neuron 2015, PMID: 25741730). The\nmechanisms underlying the splitting in hamsters, which is caused by the constant light,\nand that in mice with disturbed SCN neuronal function (AVP neuron-specific Bmal1\ndeletion, Vgat deletion, or TeNT expression), which is observable in DD, may be different.\nControl 1 Control 2\nPer1 ISH\nSampling time\nTeTN 1 TeTN 2 TeTN 3\n3-8. 5. When genetic cell manipulation was used using AAV (Figure 4, Figure 6), did you\ncheck if the results depend on the virus injection spread pattern? Was one side of the SCN\nbetter expressed by the virus compared to the other? The quality of the injection (with\nreference to Right/Left) vs. the effect should be quantified, and the interpretation should\ninclude this information.\nResponse: We checked the right/left difference of the AAV injection and presented in\nSupplementary Fig. 8 and 10 in the revised manuscript. Essentially, AAV was infected\nequally into both sides, and we failed to find the correlation between the right/left\ndifference and behavior rhythm patterns.\n3-9. 6. It is not clear to me why the initial hypothesis is that the prevention of the\nneurotransmitters' release (Figure 6) would lengthen the period. I would expect it will\nprevent cell-cell communication and will cause asynchrony, which will show\nunsynchronized LMAs at DD, when there is no entrainment. I would further claim that\nthe diversity in the results could be due to Right/Left expression of the virus. The histology\nof AAV efficiency in each mouse should be determined and presented in correlation to the\neffect.\nResponse: We always think how much easier it would be to explain if Avp-Bmal1−/− and\nAvp-TeNT mice were simply arrhythmic. However, the experimental data consistently\nindicate that this is not the case. Therefore, we proposed the SNFL model, which can\nexplain all the data, as summarized in Fig. 8. Because non-AVP cells, including VIP and\nother cells, have weak circadian oscillators with relatively longer periods on average,\nattenuated behavior rhythms with a lengthened remain even with the prevention of\nneurotransmitters’ release from AVP neurons. We called this weak oscillation\n“background circadian oscillation” or “default clock state.” We added the sentence “To\nfurther explore the evidence that AVP neurons act as the principal oscillator with an\nintrinsically short period, but its attenuation uncovers a weak, long-period circadian\noscillation of non-AVP neurons” in the Results section of Avp-TeNT mice.\nRegarding right/left asymmetry, we confirmed no significant correlation\nbetween right/left difference in TeNT expression and behavior phenotype. SCN images\nof the individual mice were presented in Supplementary Fig. 10. Of course, the variability\nof TeNT expression level and distribution may likely cause the diversity of behavior\nrhythm.\n3-10. Conclusions:\n1. At the full animal organism, other aspects should be taken into account. For example,\nwhat was the effect on melatonin? What was the core body temperature? To emphasize\nthis point, in Figure 1, the LMA in VIP-deficient #1 seems to overlap with VIP-Ca+2\nsignal after day ~20, which is very unique. It is also reflected in Figure 2c. Is it possible\nthat VIP neurons are more sensitive to entrainment, and when light entrainment is not\npresent, activity (maybe temperature) will entrain the TTFL cycle?\nResponse: We appreciate the reviewer for raising an important point. It is true that the\nVIP-Ca2+ overlapped substantially (but not completely) with the LMA in some VIP\ndeficient mice at the late stage in DD. As explained above, we are not aiming to show the\ncausal relation between weak long-period VIP-Ca2+ and behavior rhythms in VIP-\ndeficient mice (1-5). We measured VIP-Ca2+ rhythm as a representative of non-AVP\ncellular oscillators, because they were technically accessible. Moreover, the phase-setting\nand pacesetting of LMA appear partially independent of each other, as described above\n(3-3). Therefore, we would like to focus here on the pacesetting mechanism. Practically\nspeaking, melatonin is not synthesized in the C57BL/6J strain, the genetic background of\nmice used in this study, and the anticipated weak rhythm of body temperature in VIP-\ndeficient mice might be insufficient to dissect the temperature rhythm per se and the\nfluctuation due to locomotion. So, we are afraid that we cannot expect much information\nworth doubling the number of recordings from measuring these parameters.\n3-11. 2. Some critical papers are missing from the Discussion, such as a previous paper\nshowing VIP-/- activity and comparison to it\n(https://journals.sagepub.com/doi/10.1177/0748730414549767) and AVP single-cell\nimaging in vivo https://www.pnas.org/doi/epub/10.1073/pnas.2209329120\nResponse: We thank the reviewer for suggesting important papers. We cited and\ndiscussed these two papers in the revised manuscript (“Previous studies reported slight\nbut statistically significant sex differences in the behavior rhythms of VIP knockout\nmice…” in the Methods). As for the latter, please also see our response to Reviewer 2,\nwhich is too long to repeat here (“Stowie et al…” in 2-1).\n3-12. Minor:\n1. In the abstract: “VIP and other neurons occasionally exhibit weak, unstable, long-\nperiod calcium rhythms only when AVP neuronal oscillation is attenuated. ” – only when-\nnot clear. Compared to what?\nResponse: We have modified the sentence as: “only when AVP neuronal oscillation\nattenuates due to VIP deficiency or AVP neuron-specific Bmal1 deletion.”\n3-13. 2. In page 5: “In VIP-deficient mice, the AVP-Ca2+ rhythm was almost correlated\nwith the short-period behavior rhythm in DD” – this is not obvious from Figure 1. For\nexample, Figure 1d shows VIP Ca+2 offset correlates with locomotor onset. Figure 1e\nshows a significant difference between AVP-Ca+2 and locomotor activity. Please clarify.\nResponse: We appreciate the comment. We modified it as “In VIP-deficient mice, the\nperiod length of AVP-Ca2+ rhythm was comparable with the short-period behavior rhythm\nin DD.”\n3-14. 3. As for the disappearing rhythm of Ca+2 signal of VIP neurons in VIPtTA/tTA\nmice– could it be due to VIP neurons' apoptosis? As VIP is deficient in these mice, I would\nsuggest that the neurons had some decline in their health, especially when combined with\nGCaMP, which is toxic. Did you check VIP neuronal health after imaging?\nResponse: We agree that this point is important. We compared the number of jRGECO1a-\nexpressing cells between heterozygous and homozygous as an indicator of VIP neuronal\nhealth in the revised manuscript, which turned out to be comparable (Supplementary Fig.\n1e).\n3-15. 4. It is not clear how Figure 1C and 1F include VIP Ca signal for 25 days, but the\nanalysis in d and e is only up to day 14.\nResponse: In Fig. 1, we characterized the behavior rhythms by actograms and Ca2+\nrhythms by the curve lines of detrended, smoothened jGCaMP7s/jRGECO1a signals, as\ndescribed in the Methods. As the reviewer pointed out, we gave up calculating the values\nfor VIP-Ca2+ after a week in DD because it became too weak and noisy to analyze by this\nmethod accurately. In Fig. 2, we ran periodogram analyses to examine their periodicity\nobjectively and quantitatively. We further clarified the procedure in the manuscript\n(Results, Methods, and Legends).\n3-16. 5. Why Figure 1 and 2 are separated? They both show the same experiment with\ncontinuous analysis approaches.\nResponse: We just thought that including all panels of Figs. 1 and 2 in one figure would\nmake the individual panels too small to see. We used different analyses of the same\nrecorded data for Figs. 1 and 2. If the reviewer thinks that these two figures should be\ncombined into one figure, we would be grateful to follow the instruction.\n3-17. 6. Based on Figure 1 and Supp Figure 1, some mice had a shift towards ~24h rhythm\nafter day ~18, and some did not. Did you try to analyze the cohort separated into two\ngroups, based on this observation? Do you think it can be predicted based on a feature in\ntheir LD activity?\nResponse: Indeed, we initially considered separating VIP-deficient mice into two groups\nas the reviewer suggested. However, as the periodograms in Supplementary Fig. 2 show,\nwe encountered a difficulty in setting a clear border to separate one peak from two peaks.\nTherefore, we gave up doing so and presented all the individual data. We failed to see\nclear correlation between the LMA patterns in LD and DD.\n3-18. 7. Figure 6: E and M should be indicated in Figure 6c.\nResponse: We have indicated E and M activities in the actograms presented in\nSupplementary Fig. 10c.\n3-19. 8. In the Discussion: each of populations - > each one of the populations / each\npopulation\nResponse: We have corrected it as the reviewer pointed out.\n3-20. 9. In the Discussion: “Nevertheless, this method is so far the only one that allows\nlong-term recordings of SCN dynamics in genetically defined neuronal populations in\nvivo.” – this sentence is not correct; see, for example,\nhttps://www.pnas.org/doi/epub/10.1073/pnas.2209329120\nResponse: We discussed and compared the pros and cons of this reference (Stowie et al.,\nPMID: 36656857) and our current study in the second paragraph of the Discussion. Please\nalso see our response to Reviewer #2 (2-1).\n3-21. 10. Pg. 13- SNFL - not defined\nResponse: We defined the SNFL in the last section of the Results.\n3-22. 11. While the paper did not mention differences between males and females, this\nshould be considered, given the differences found here:\nhttps://www.biorxiv.org/content/10.1101/2022.08.18.504454v2.abstract\nResponse: We mentioned and cited the sexual difference in the mouse behavior\n(“Previous studies reported slight but statistically significant sex differences in the\nbehavior rhythms of VIP knockout mice…” in the Methods). We also confirmed that our\nconclusion did not change when male and female mice were analyzed separately (e.g.,\nSupplementary Fig. 3). The sex of all mice used in this study was declared in the\nsupplementary figures and the source data file.\nResponse to Reviewers\nWe thank the reviewers for their constructive comments and suggestions, which have\nsubstantially improved the quality of our manuscript. The manuscript has been revised\naccordingly, and our point-by-point responses to the reviewers’ comments are provided\nbelow.\nResponse to Reviewer #1\nI commend the Authors for their thorough responses to my comments.\nMy concerns/ reservations have been accurately addressed.\nResponse: We appreciate the reviewer’s positive comments.\nThere is one minor point:\nNevertheless, to avoid such a concern, we also analyzed with multiple time windows\nwithout overlap and reached the same conclusion (Supplementary Fig. 1f,g). If the\nreviewer prefers this presentation, we would be happy to replace the graphs.\nI do prefer this method of analysis and so I think it should replace the original plots.\nResponse: We replaced all applicable graphs with versions that did not overlap in time\nwindows (Fig. 2d and 2e, Fig. 3e, Fig. 4g and 4h, Supplementary Fig. 1a, Supplementary\nFig. 2, Supplementary Fig. 4, and Supplementary Fig. 5). This revision does not change\nour conclusions. One minor difference is the loss of statistical significance in Fig. 3e. The\ndisappearance of significance is likely due to the large variability among individual\nmice’s actograms and the use of 5-day windows, which may be too short for precise\nperiodogram analyses as noted in the main text. Nevertheless, the other analyses in Fig.\n3b and 3d clearly show that the two groups differ significantly in their free-running\nperiods. Furthermore, the purpose of Fig. 3 is to demonstrate that Avp-CK1δ−/−; ViptTA/tTA\nmice—unlike VIP-deficient mice with intact CK1δ—never initiate their free-running\nrhythms with a short period of approximately 22.6 h. Thus, the revision does not affect\nthe conclusions of our study.\nIn my opinion, they have also done an excellent job of responding to the other Reviewers'\ncomments.\nResponse: We are very grateful for the reviewer’s comments to date, which have guided\nthe manuscript toward a substantially improved form.\nResponse to Reviewer #2\nI thank the authors for a well-reasoned and thorough response to prior review. I maintain\nthat this manuscript represents a technical marvel, but the impact of the results have a\nlimited scope, and don't do much to substantially further our understanding of this circuit.\nResponse: Thank you for the reviewer’s positive assessment that we have appropriately\naddressed the initial review. We also appreciate the reviewer’s high evaluation of the\ntechnical aspects of this work. While we respectfully acknowledge the reviewer’s\nassessment that the manuscript provides limited advancement in scientific understanding,\nwe take the reviewer’s comments seriously and will use them to guide our future research.\nResponse to Reviewer #3\nOverall, the authors have answered my questions from the first round of review.\nThe writing improved, and the overall message is much clearer.\nThe addition of the model was very informative and went beyond my expectations. I think\nthe model does a great job of explaining the results, and the supplementary materials are\nwell-explained.\nResponse: We appreciate the reviewer’s positive comments. We also thank the reviewer\nfor their initial comments, which led to substantial improvements in the manuscript.\nMinor:\nIn the Abstract\n“Meanwhile, VIP amplifies and delays the AVP neuronal rhythm daily, thereby\nlengthening its period to approximately 24 hours in constant darkness”\nThis sentence comes after the sentence about using VIP deficient mice – so it is not clear\nwhere the VIP comes from, and the connecting word “meanwhile” is confusing.\nResponse: We thank the reviewer for pointing out this critical issue. We have revised the\nsentences as follows: “Here, by monitoring cellular calcium rhythms in vivo using dual-\ncolor fiber photometry in mice lacking vasoactive intestinal polypeptide (VIP), we\ndemonstrate that arginine vasopressin (AVP) neurons oscillate intrinsically with a short\nperiod and reduced amplitude. This indicates that VIP normally amplifies and phase-\ndelays the AVP neuronal rhythm each day, thereby lengthening its period to\napproximately 24 hours in constant darkness.” We also modified the text to reduce the\nword count.\nRegarding the model, can it include a compensation mechanism that explains the results\nin Figure 4?\nResponse: We thank the reviewer for the valuable suggestion. We believe that the\nreviewer is referring to the compensatory mechanism implicated in Figure 5. To address\nthis point, we added the following sentence to the Results section explaining the SNFL\nmodel: “In addition to this primary direct pathway, there may also be minor indirect routes\nthrough which VIP regulates AVP neurons via other SCN cells (Fig. 5),” and modified the\nright panel of Fig. 8a accordingly. We also cited Fig. 8a in the Discussion: “Therefore, it\nis tempting to speculate that the indirect, redundant, and compensatory pathways play a\nrole in regulating the AVP neuronal period by VIP to tightly control the SCN ensemble\nperiod (Fig. 8a).”\nThe code is clear and well annotated\nResponse: We appreciate your positive evaluation."}]

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