Estrous Cycle and Egg Release Resets with Light Entrainment Inspired by Neurodynamics in the Circadian Pacemaker

doi:10.21203/rs.3.rs-2069847/v1

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Estrous Cycle and Egg Release Resets with Light Entrainment Inspired by Neurodynamics in the Circadian Pacemaker

https://doi.org/10.21203/rs.3.rs-2069847/v1

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Jet lag, shift work, and the extended light hours of modern life disrupt the regular light-dark cycle, affecting the menstrual cycle and decreasing fertility. The circadian pacemaker, the suprachiasmatic nucleus (SCN), is known to modulate ovulation, but the exact mechanism is not understood. Here we explore this connection by imaging neuronal activity in freely-behaving mice following extrinsic (jet lag) and intrinsic circadian circuit perturbations. We show that retina-connected vasoactive intestinal peptide (VIP)-expressing neurons in the SCN directly relay light information to gonadotropin-releasing hormone (GnRH)-expressing neurons to gate ovulation, as measured by recording calcium-sensitive activity of SCNVIP neurons over days in freely behaving mice. We show with machine-learning-based discriminations that the neuronal light-synchronized activity is primarily sex-dependent, and estrous-dependent in the late afternoon. Consistent with such a light-driven switch, we find that a pulse of light in this critical period could rescue estrous cycle regularity and the number of eggs released by animals kept in limited-light conditions. Our in vivo approach thus reveals the dynamic mechanism by which SCNVIP neurons trigger ovulation and demonstrates light-induced rescue of fertility, a finding with potential therapeutic implications for humans struggling with infertility due to shift work or jet lag.

Estrous cycle

suprachiasmatic nucleus

vasoactive intestinal peptide

circadian rhythm

GnRH

calcium imaging

fiber photometry

CRISPR

In the mammalian estrous cycle, fluctuations in sex hormone secretion occur with periods longer than a day, and these infradian rhythms are coordinated with circadian rhythm to align the fertility window with social interaction and receptivity, thus maximizing reproduction¹. The critical event of ovulation is controlled by the hypothalamic-pituitary-gonadal (HPG) axis and, in mice, is synchronized with the dark phase every four or five days. In metestrus and diestrus, estrogen secretion from the ovary increases, followed by a surge in luteinizing hormone (LH) secretion in the late afternoon of proestrus, triggering ovulation ~12 hours later. Hypothalamic neuronal populations such as gonadotropin-releasing hormone (GnRH) neurons in the medial preoptic area (MPA) ², RF-amide-related peptide-3 (RFRP-3) neurons in the dorsomedial hypothalamus (DMH) ³ and kisspeptin (KISS1) neurons in the anteroventral periventricular nucleus (AVPV) ^4,5 and the arcuate nucleus⁶ mediate estrogen feedback and are responsible for the LH surge.

The hypothalamic suprachiasmatic nucleus (SCN), the circadian rhythm pacemaker, is fundamental to ovulation occurrence. Several pieces of evidence suggest that signals from the SCN directly generate the LH surge. First, the SCN has neuronal projections to relevant hypothalamic regions, including the MPA ⁷, AVPV ⁸, and DMH ^8-10. Second, SCN lesions cause a decrease in LH surge and estrous cycle regularity ^11-14. Two circadian neuropeptides have been associated with the LH surge: arginine-vasopressin (AVP) and vasoactive intestinal polypeptide (VIP). Female mice deficient in VIP show reduced estrous-cycle regularity and fertility ¹⁵, and neurons expressing these peptides project directly or indirectly to GnRH ^16-18, Kiss1 ⁶, and RFRP-3 ¹⁰ neurons. Specifically, VIP receptor type 2 (VPAC2, encoded by vipr2) is present in ~40% of GnRH neurons during proestrus, and VIP processes have been observed in apposition to GnRH neurons ^7,19. Based on bilateral differences in c-Fos expression in GnRH neurons in hamsters, it was suggested that circadian regulation of GnRH neurons is mediated by direct synaptic input to GnRH neurons ²⁰. While VIP is known to excite GnRH neurons on brain slices, the estrous cycle regulation of this process is debated in the literature ^16,18,21, and the temporal activity pattern of VIP-expressing neurons along the estrous cycle is unknown.

Here, we show that light information affects estrous cycle regularity through a direct path from SCN^VIP neurons to GnRH neurons via VPAC2. Measuring the dynamics of these neurons across the estrous cycle using calcium indicator with fiber photometry, along with sensitive machine-learning-assisted signal analysis, we show a sex-specific, light-active switch that provides a circadian, light-driven input to GnRH neurons. This finding directly inspires a light exposure paradigm capable of rescuing estrous cycle regularity and oocyte quantity in near-constant-dark conditions. Together, this work shows the strong mechanistic connection between light, SCN^VIP activity, estrous cycle, and fertility.

Estrous cycle regularity in mice is significantly reduced by jet lag or ablation of SCN^VIP neurons

In humans, disruptions in the regular light-dark cycle (e.g. during “jet lag”) have been associated with irregular menstrual cycling and decreased fertility. To confirm the significance of light regularity on the mouse estrous cycle, we performed a “jet-lag” experiment in which, every 4-5 days, we advanced the light in the room by six hours, which is equivalent to flying from New York to Honolulu, four days later to Hong Kong, followed by Zurich and back to New York, all in three weeks. This specific pattern was previously found to dramatically decrease pregnancy success in mice [33], and reduce fertility in human in general ^22,23. Using cytology of daily vaginal smears, we found that this light pattern decreased the regularity of the estrous cycle in C57 female mice, reducing the number of estrous cycles from 3.6±0.4 to 1.9±0.2 (mean±sem) in three weeks. This effect was due mainly to an increased time spent in estrus, from 7.0±0.4 to 9.1±0.8 days (Figure 1A-C).

The SCN is one of the first brain relays for retinal responses to light. SCN^VIP neurons were previously identified as essential to the circadian response to light ^24-28, forming monosynaptic connections to intrinsically photosensitive retinal ganglion cells (ipRGCs) ^26,29 and mediating the rapid response to light during the dark phase ²⁵. We therefore tested the effect of specific ablation of SCN^VIP neurons on estrous cycle regularity. We injected adeno-associated virus (AAV) to deliver caspase-3 to the SCN of VIP-Cre mice, inducing targeted apoptosis of SCN^VIP neurons (Figure 1D, E). We found that ablation significantly reduced the number of estrous cycles, from 4.3±0.4 to 2.8±0.3 in three weeks (Figure 1F). These results support a specific role for SCN^VIP neurons in connecting light stimulus with estrous cycle regulation.

VIP acts directly on GnRH neurons through VPAC2 to regulate proestrus regularity

The specific SCN^VIP ablation results could be due to either direct release of VIP to GnRH neurons or an indirect effect of local SCN interactions, such as with AVP neurons, which further project to Kiss1 neurons ^30,31, the pulse generator for GnRH neurons ³². To test this, we used CRISPR genetic editing to mediate disruption to VPAC2 expression in MPA^GnRH, and tracked estrous cycle regularity. In a separate cohort, we disrupted the expression of Kiss1 receptor (KISS1R). To specifically target GnRH neurons that project to the median eminence (ME), we used AAVretro injected into the ME for delivery ³³ (Figure 2A). Female mice given a control injection exhibited a regular estrous cycle, with an average frequency of five estrous cycles in three weeks. CRISPR-mediated knockout approach of either kiss1r or vipr2 resulted in a significant reduction in the number of estrous cycles (from 4.2±0.3 to 2.7±0.3, p=0.027, in sgKiss1R and from 4.8±0.3 to 2.8±0.3, p=0.007, in sgVipr2, Figure 2B). We verified the disruption to the receptor expression by labeling (Figure 2C) and co-expression (Figure 2D), which showed that even low injection efficiency led to a significant physiological effect. These results provide strong evidence that VIP neuropeptide has a direct effect on GnRH neurons through VPAC2.

Circadian temporal dynamics of SCN^VIP are sex-dependent and not estrous-cycle dependent

Prior work with slice electrophysiology showed that VIP increases the activity of GnRH neurons in a time-of-day dependent manner ^16,18. To learn how the dynamics of SCN^VIP neurons affect GnRH neurons in vivo in response to circadian and hormonal feedback, we used fiber photometry (FP) to record long-term bulk GCaMP6s activity for 10 minutes each hour over multiple days in VIP-Cre x GCaMP6s mice, including males and both naturally cycling and ovariectomized (OVX) females (Figure 3A). As expected, we observed day-night fluctuations in both sexes, with reduced activity during the dark hours between Zeitgeber (ZT)12 and ZT24 (Figure 3B, C). SCN^VIP neurons showed high median values of integrated dF/F and event rate signals during the light phase and low dF/F calcium signals during the dark phase (0.35±0.02 and 0.05±0.02 events/min and 6±1 and 2.5±0.3 a.u. integrated dF/F during light and dark phases respectively). We observed that females had higher integrated dF/F compared to males but no difference in event rate (8.7±0.8 and 3.5±0.5 a.u. integrated dF/F, and 0.36±0.02 and 0.34±0.02 events/min, females and males respectively, Figure 3C). A significant difference in event rate appeared between males and females in ZT1 (0.35±0.07 vs. 0.52±0.07 events/min, males vs. females), and in dF/F during most light hours (Figure 3D). These differences suggested a role for sex hormones on SCN^VIP neurons.

Next, we tested the dependency of SCN^VIP neuronal activity on the estrous cycle. We combined vaginal smear cytology with SCN^VIP FP recordings of cycling females and compared them to OVX females' activity. To be more accurate with the estrous cycle days, we termed the days along the estrous cycle as P-2 to P+2, which refers to P+0, which is the proestrus day. Typically, P-2 and P-1 are metestrus or diestrus, and P+1 is estrus. P+2 is estrous and metestrus (see also ³⁴). We did not identify significant differences between estrous stages or between intact and ovariectomized females (Figure 3E, see also Figure S3). Comparing more limited time windows, we observed higher event rates in P-1 than P+1 during the late afternoon (ZT11-12), but none of the comparisons showed a significant difference between estrous days or OVX (Figure 3F). We did, however, observe significantly increased event rates during the light phase for all estrous stages (0.36±0.04 vs. 0±0 events/minute, light vs. dark, averaged over estrous stages), and increased dF/F values for some (for example, 7.3±0.8 vs. 2.1±0.1 a.u. for light vs. dark in P-1, Figure 3G). These FP data suggest that the main role of SCN^VIP neuronal activity, which is sex-dependent but lacks estrous-cycle dependency, is a circadian switch aligned with light information to convey the best fertility timing.

Given the dramatic hormonal changes (producing the LH surge) that occur during ZT11-12 of the proestrus day, we more closely examined this time window using fast Fourier transform (FFT) analysis. FFT transforms data from the time domain to the frequency domain, revealing detailed information about possible contributing oscillators ³⁵. We combined FFT with a machine learning classifier to test whether it could distinguish SCN^VIP activity patterns between males, OVX females, and females at different days in the estrous cycle. We applied FFT to each 24h recording session described above, yielding power spectra for different frequency ranges (Figure 4 A-C). Autocorrelation of these individual integrated power spectra over days revealed a high amplitude 24h rhythm for two frequency ranges (0-0.03, 0.03-0.5 Hz), and a low amplitude third (0.5-1.6 Hz), and did not show a unique feature for the proestrus day (Figure 4D). We then tested finer frequency ranges, creating a spectrogram representing frequency contributions to different states (Figure 4E). To test whether patterns are informative at the population level, we averaged these FFT matrices over animals and states (Figure 4F) and compared P-1 vs. P+1 and OVX vs. male in the critical ZT9-12 window (Figure 4G-J). At lower frequencies (0-0.03 Hz) at ZT11 and ZT12, FFT separated OVX and male animals, but not P-1 and P+1 females (ZT11: 31±3 a.u. (median±sem) vs. 17±3 for OVX vs. male, and 34±3 vs. 37±3 for P-1 vs. P+1; ZT12: 42±4 vs. 19±3 for OVX vs. male and 36±4 vs. 45±5 for P-1 vs. P+1). We found similar results comparing ZT9 and ZT10 (Figure S2, A-D). Finally, we tested whether a support vector machine (SVM) classifier could discriminate estrous states or sex. For simplicity, we used leave-one-out cross-validation (LOOCV) approach (see Methods, Figure 4K, 0.5 indicates the chance level). Within female estrous states, the cross-validation score was about the chance level except for P+1 vs. P+2 (61±2% prediction accuracy). Even simplifying estrous cycle days to receptive and non-receptive did not enable classifier discrimination (51% accuracy). A reduced-dimension spectrogram comprising only ZT7-12 (Figure S2 E), and an alternative (“Discriminate analysis”) algorithm (Figure S2 F) gave similar results. These results support the idea that SCN^VIP activity shows sex-specific patterns but is not differentially regulated through the estrous cycle.

SCN^VIP activity peaks at ZT10 in the days before proestrus

Given the unique SCN^VIP GCaMP signal we observed during the late afternoon, at ZT11-12, we decided to continuously record FP data around this time to identify transient changes associated with the LH surge that we might have missed by recording only 10 minutes per hour. We recorded continuously for two hours, ZT 11-13, and compared males and females, as well as from ZT 10-10.5, the approximate time of LH onset during proestrus, and compared females at different estrous stages. The comparison between males and females at ZT11-12 revealed higher event rates during the light phase during ZT 11-12 compared to ZT 12-13 in females but not in males (Figure S4, also showing dark to light transition, ZT 23.5-0.5, for comparison). We simplified females’ estrous stages into receptive (RE) and non-receptive (NR), based on estrogen and progesterone profiles ³⁶. Previous findings have shown that the SCN is estrogen-sensitive ^37-39, and therefore we hormonally induced OVX females with estrogen (OVX+E) followed by progesterone (OVX+E+P) to detect the effects of stimulated proestrus (see Figure S5 for full estrous-cycle analysis). Analysis revealed decreased event rates during receptive days, with hormonal stimulation of OVX females insufficient to rescue the receptive activity pattern (Figure 5E, F).

We next applied our FFT-SVM classification approach to test whether the estrous state can be discriminated based on SCN^VIP activity at ZT 10-13. We created spectrograms by dividing 150 minutes of recording into ten-minute intervals (Figure S5, averaged spectrogram shown in Figure 5H). Classification using the ZT10-10.5 FFT matrices of females could discriminate above chance level between P-2 and receptive days (53-59%), as well as between P+2 and P-1 (65%). To assess hormonal stimulation, we compared RE, NR, OVX and hormone-stimulated (OVX+E) females. Discrimination ability between RE and NR remained slightly above the chance level (58±1%), while both OVX and OVX+E could be discriminated from both RE and NR with better than 60% accuracy, suggesting that estrogen alone cannot rescue the activity patterns of cycling females, at least under these conditions. Together, our results show a significant difference in SCN^VIP neuronal activity at ZT10 across the estrous cycle.

Afternoon SCN^VIP activation is not sufficient to rescue estrous cycle regularity

Since SCN^VIP neurons showed increased activity during daytime, with dependency on the estrous cycle at ZT10, we hypothesized that SCN^VIP activation during the afternoon could rescue the irregular estrous observed in DD conditions to sufficiently stimulate GnRH neurons and induce ovulation. We used two genetic approaches: Chemo- and optogenetic activation using an excitatory DREADD (hM3Dq) or ChR2. Both methods have been established previously for SCN^VIP neurons ^25,40-42; however, the chemogenetic approach induces calcium flux with a single injection of clozapine N-oxide (CNO), a synthetic drug used mainly in biomedical research as a ligand to activate DREADD receptors, and produces longer time scales of activation, which might better represent the natural activation of SCN^VIP neurons. To test this, we assayed VIP-Cre females’ estrous cycle under four conditions: LD, DD with 0.5 h of light at circadian time (CT) 0 (DD + L0), and DD+L0 with an additional hour of light either prior to the expected LH surge, at CT10, or at an earlier time point in the day, at CT4, as a control (DD+L0+C4 or C10, Figure S6). 0.5h of light in DD conditions was sufficient to preserve the LD locomotor activity (LMA) profile (Figure S7). We found that afternoon activation (DD+L0+C10) of the hM3Dq expressing group caused a trend (not statistically significant) of increased estrous cycles. We verified CNO activation post hoc by c-Fos expression (Figure S6). In addition, we activated SCN^VIP neurons in VIP-Cre x ChR2 mice at similar patterns. Our results showed that ChR2 activation at CT10 was not significantly different from both control groups (VIP-Cre x GFP with blue light excitation or VIP-Cre x ChR2 with no excitation). We verified optogenetic activation and fiber localization using a 3D histological method that we developed to allow post hoc investigation of the precise positioning of optical implants within deep and small brain nuclei, such as the SCN, named light-guided-sectioning (LiGS) ²⁴ and verified the activation of SCN^VIP neurons using c-Fos labeling under the fiber (Figure S8). These results indicate that either different activation patterns of SCN^VIP or other circuits are required for light-induced estrous cycle regulation.

Afternoon light rescues estrous cycle regularity and egg release under limited light conditions

Understanding that the effect of afternoon light cannot be minimized to a particular cell type in one site ⁴³, we suspected that a positive effect of light on fertility could come with a careful design of the light stimulus at the in vivo full physiology level, and not only by SCN^VIP activation, but inspired by its dynamics. We used the same light conditions as in the previous experiments (Figure S6, S8). In the LD cohort, we observed an average of 4.2±0.5 estrous cycles in three weeks, indicative of a normal estrous cycle. DD and DD+L0 conditions reduced this to 2.8±0.8 and 1.8±0.3 estrous cycles in three weeks, respectively, consistent with the SCN^VIP specific activation experiments (Figure S6, S8). One hour of light at CT10 was sufficient to rescue the regularity of the estrous cycle; we measured 4.8±0.3 estrous cycles in three weeks, significantly more than with light stimulation early in the day at CT4 (Figure 6B). The DD+L0+L10 condition also rescued the general estrous cycle profile (Figure 6C). While estrous cycle regularity in itself can indicate fertility, we directly tested the effect of light manipulations on fertility outcome by measuring the number of eggs released to the oviduct on the day following ovulation (estrus). We avoided using pregnancy success as a fertility readout since pregnancy itself might involve circadian clock cues ⁴⁴. We observed a significantly increased number of oocytes released in response to the rescue pattern of light (DD+ L0+ L10), with approximately twice the number of eggs released as in the DD+L0 condition (Figure 6D). Together, these results show that light manipulations reduce estrous cycle regularity and that light in the late afternoon is critical for both estrous cycle regularity and to increase egg release.

Here we find that estrous cycle regularity in mice is significantly reduced by either jet lag or ablation of SCN^VIP neurons. We establish a specific and direct role for VIP neuropeptide signaling to GnRH neurons through its receptor, VPAC2, and find significant differences in calcium dynamics of SCN^VIP neurons between males and females. Using power-spectral analysis, we find a significant reduction in event rates during receptive days at ZT10, indicating a hormonal dependence. This observation led us to hypothesize that a pulse of afternoon light would rescue estrous cycle regularity in limited light conditions, as indeed we found. To our surprise, the number of released eggs also increased, indicating a significant improvement in fertility. While a collection of released eggs is not commonly performed in naturally cycling mice, our close monitoring of the estrous cycle allowed us to do so. We found this information critical, as it suggested a strong coupling between light cues and follicular maturation without introducing the additional hormones required for superovulation ⁴⁵.

The decrease in ovulation we observed is aligned with estrous cycle regularity reduction in response to different light-dark schedules ⁴⁶, and other dark-dark (DD) experiments ^47,48. It was previously shown that a full-body VIP-KO female mouse had reduced estrous cycle regularity ¹⁵. By monitoring the estrous cycle alongside viral manipulations, we show that physiological fertility is specifically effected by VIP neurons localizes at the SCN and VIP receptor on GnRH neurons. Previous studies established the VIP-GnRH connectivity on brain slices, showing that VIP excites GnRH neurons ^16,18, and reporting anatomical connectivity ^7,17,19; while it is possible that other VIPergic populations might signal to GnRH neurons through VPAC2 ⁴⁹, our results of VPAC2 expression-disruption confirm the main findings regarding VIPergic control on GnRH neurons to result in a perturbation effect on ovulation with full body physiological outcomes.

In GCaMP experiments, we measured the bulk activity of SCN^VIP neurons. One benefit of bulk fiber photometry measurement is that it was sufficiently powered for a machine learning (ML) algorithm to predict sex and estrous state based on power-spectral analysis of neuronal activity. ML is currently used to identify sex in biological samples or behaviors ^50-53, and, while ML has been used to identify estrous cycle stages in vaginal smears ^54-56, this is, to our knowledge, its first use to identify estrous cycle states based on neuronal activity.

The dependency of SCN^VIP neuronal activity on sex and hormone levels raises the question of its mechanism. Interestingly, in addition to the estrogen dependency mentioned above ^24,37-39, hypothalamic single-cell RNAseq data suggest that VIP-targeted neurons have progesterone receptor (PR) mRNA, and AVP neurons appear to coexpress Esr1 ⁵⁷. It will be interesting to test whether VIP and AVP neurons co-localize with PR and Esr1 in situ. Surprisingly, we did not observe receptive-like activity in the hormonal stimulation paradigms; it could be that a long period following ovariectomy without a hormonal substitute caused a loss of plasticity that brief hormonal treatment could not rescue.

While our work focuses on the direct role of SCN^VIP neurons on GnRH neurons, a role for AVP neurons cannot be ruled out, and future work could focus on the interaction between VIP and AVP neurons and the unique role of AVP dynamics on GnRH and Kiss1 neurons. We should also note that SCN^VIP neurons are not homogenous in at least two aspects; First, SCN^VIP neurons release two neuropeptides, VIP and GABA ⁵⁸. Jones et al. ²⁵ showed that SCN^VIP neurons' calcium activity correlates with VIP release, so we examined the role of VIP, but we cannot rule out a role for GABA as well. Second, recent studies have functionally ⁴¹ and genetically ^26,59 defined two subpopulations of SCN^VIP neurons, and it will be interesting to address subpopulation-specific differences in the future.

Based on our results, we suggest the following model : the time-of-day-dependent oscillating activity of SCN^VIP neurons ^25,60 gives constant input to MPA^GnRH neurons, mediated by VIP release and VPAC2 expression on GnRH neurons. As estrogen levels go up (prior to ovulation), GnRH neurons, which do not express estrogen receptors, increase activity by integrating information from SCN^VIP neurons (indicating ‘day’), with that from inhibitory DMH^RFRP-3 neurons ¹⁰ and excitatory Kiss1 neurons ⁶¹, both of which express estrogen receptors. Following GnRH release and LH surge, estrogen levels drop, and progesterone levels rise. Based on our recording data, these hormonal changes cause a decrease in SCN^VIP neuronal activity at ZT10 to release less VIP to GnRH neurons during proestrus afternoon and estrus, to prevent the reoccurrence of ovulation (Figure 7). When the light information is disrupted or reduced, less VIP is secreted and MPA^GnRH activity cannot be elevated as needed. However, the accurate timing of afternoon light is enough to cause a release of VIP (and possibly other neuropeptides) to rescue GnRH neuronal activity and fertility outcomes. Our opto- and chemo-genetic activation of SCN^VIP neurons alone was insufficient to rescue the phenotype, indicating that the effect of light is physiological and beyond the level of one molecular cell type at one brain site.

In an era of ever-increasing light pollution due to artificial light, and with increasing evidence that shifted working hours are associated with reproductive disorders in humans ⁶², our work in mice offers a temporal approach to study the HPG axis and its neuronal control by the circadian rhythm pacemaker. In particular, our observation of increased egg release in response to targeted light intervention may inspire potential interventions for women who struggle with infertility due to shifted working hours or continuous jet lag ^63-65.

Experimental animals

Mice used in this work include C57BL/6NCrl (Charles River), VIP-IRES-Cre (Jackson Laboratory Stock, JAX, 010908) crossed to C57BL/6NCrl (Charles River), or Ai162 (GCaMP6s reporter line, JAX, 031562). Doxycycline diet was used to suppress expression until 2-4 weeks before surgery. VIP-IRES-Cre line was crossed to Ai132 (ChR2, JAX, 24109). GnRH-Cre line (Gnrh1-Cre, JAX, 21207) was crossed to Rosa26-Cas9 (JAX, 24858). Animals were group housed (2-4 per group) whenever possible to ensure a regular estrous cycle. Mice were single-housed for FP experiments that required 24/7 recordings and optogenetic experiments. Females that were single housed for calcium recording received a handful of fresh male mice bedding every cage change to ensure regular estrous cycle. Mice had a running wheel or a cleared tube for enrichment in their home cage. For optogenetics, mice were connected with two-branched patch cables, so running wheels/tubes were impossible. Mice were kept at 12:12 light cycle, unless stated otherwise. Mice were used until 10 months old to ensure a regular estrous cycle ⁶⁷, and were age-matched for each experiment. Animals had ad libitum access to food and water.

FP recording, light manipulation, optogenetic and chemogenetic experiments were performed in rat cages (40 x 34 cm). For FP, the lid was modified to have a hole in the middle for the patch cable.

Light manipulation experiments with PIR detection and/or optogenetic manipulation were performed in rat cages, with a 10-inch diameter Plexiglas cylinder, 34 cm in height, to ensure sufficient PIR detection. Mice were group housed for light manipulation experiments. For optogenetic experiments, mice were single-housed due to the patch cable. Other light manipulation experiments (cohort 1 of DREADD, C57) were performed in regular mice cages.

Animal husbandry and experimental procedures involving animal subjects were conducted in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Institutional Animal Care and Use Committee (IACUC) and by the Office of Laboratory Animal Resources at California Institute of Technology under IACUC protocol 1739. Mice were excluded from the entire FP experiment if there was no dynamic photometry signal or no two-photon signal 3 or 5 weeks after surgery, respectively.

Surgery

Stereotactic viral vector injections were made in mice anesthetized with isoflurane (5% induction, 1–1.5% maintenance) and placed on a stereotaxic frame (942, David Kopf Instruments, CA, USA). An incision was made to expose the skull, including Bregma, lambda, and the target sites’ external coordinates. Stereotaxic coordinates were measured from Bregma and were based on the Mouse Brain Atlas ^68,69, and improvement was made based on 2D or LiGS histology. A craniotomy hole was drilled above the target. Virus injection and implantations were performed as follows:

Cell apoptosis: VIP-Cre mice were injected with AAV5.Flex.taCasp3.TEVP virus (Experimental, 2.9E12 VG/ml) or AAV5.Ef1a.DIO.eYFP.WPRE.hGH (control, 2.3E12 VG/ml), AP -0.3/-0.2 mm, ML ± 1.19 mm, DV -5.75 and -5.6 mm from the brain, left and right sides, at 13 degrees, 2 x 350 nl each side.

Excitatory DREADD: VIP-Cre mice were injected with excitatory DREADD or control virus at the SCN; AAV5.hSyn.DIO.hM3D(Gq).mcherry (1.0E13 VG/ml) or AAV5.Ef1a.DIO.eYFP.WPRE.hGH (1.0E13 VG/ml) respectively, AP -0.3/-0.2 mm, ML ± 1.19 mm, DV -5.75 and -5.6 mm from the brain, left and right sides, at 13 degrees, 2 x 300 nl each side.

Genetic ablation in GnRH neurons: GnRH-Cre x Cas9 mice were injected with AAVrt.U6-sgRNA(SapI)-CAG-mRuby2-WPRE-hGHpA (1.4E13 VG/ml) or AAVrt.CAG.flex.tdTomato (6.4E12 VG/ml, control), into the median eminence, targeting GnRH processes ³³, prepared as described in the following section. AP -1.4 mm, ML ± 0.3 mm, DV -5.65 and -5.4 mm from the brain, left and right sides, at 0 degrees, 2 x 300 nl each side.

All viruses were injected at a rate of ~80 nl/min using a blunt 33-gauge microinjection needle within a 10 µl microsyringe (NanoFil, World Precision Instruments, WPI) by an UltraMicroPump (UMP3-4, WPI), controlled by a pump controller (Micro4, WPI)

Fiber photometry (FP), Optogenetics: Two optical fibers with a cut length of 7 mm and diameter of 400 μm (MFC_400/430-0.48_7mm_ZF1.25_FLT ,NA=0.48, Doric lenses, Canada) were firmly mounted to a stereotaxic holder. Two fibers were implanted to improve the probability of successfully targeting the structure. A thin layer of Metabond (Parkel) was applied to the skull surface to secure the fiber. In addition, a thick layer of black dental cement (JET denture repair powder and liquid) was applied to secure the fiber implant and to prevent interferences between the excitation light to light-sensitive targets which are not the SCN.

Extended details about viruses and implants can be found in Table S1.

In some cases, following implant surgery, an ovariectomy (OVX) was performed, as follows:

OVX: Each mouse was given a single dose of ketoprofen 5 mg/kg SC and sustained-release buprenorphine at 1mg/kg SC. The mouse was then anesthetized with 1-5% isoflurane in an induction box followed by maintenance in a nose cone and was maintained on a heating pad throughout the surgery. A small dorsal midline incision was made over the abdomen. The abdominal cavity was entered via a blunt puncture through the abdominal wall. The ovary was dissected. The fat pad and tissue were returned to the abdominal cavity, and the abdominal wall was closed with 4-0 absorbable multifilament suture in an interrupted pattern. The process was repeated on the opposite side through a single incision. The skin incision was closed with surgical wound clips or by suturing with a monofilament 4-0 suture material in an interrupted pattern. Before closure, bupivacaine (1 mg/kg of 0.25% solution) was applied subcutaneously to the wound margins. Mice received 30mg/kg Ibuprofen ad lib (20 mg per 100 ml water) for at least 5 days. For all OVX females, surgery success was verified by collecting vaginal smears for at least 10 days, showing either diestrus or metestrus states.

All mice were given 1 mg/kg sustained-release buprenorphine and 5 mg/kg ketoprofen s.c. intraoperatively and received 30 mg/kg ibuprofen p.o. in their home cage water for at least five days postoperatively for pain. Mice were allowed a minimum of 14 days for surgical recovery before participation in behavioral studies.

Genetic knockout in GnRH neurons using CRISPR

Guide RNA cloning and validation: sgRNA sequences were validated using pSpCas9(BB)-2A-GFP (PX458; a gift from Feng Zhang, Addgene ID: 48138). Briefly, Neuro-2a cells (ATCC CCL-131) were transfected in duplicate with PX458 containing sgRNAs of interest, and their DNA was extracted at 72 hours post-transfection (QuickExtract DNA Extraction Solution, Lucigen, QE09050). The targeted region was amplified with PCR, and Sanger sequenced. Reads were analyzed for editing efficiency using ICE analysis (Synthego). Only sgRNA sequences that yielded >70% editing efficiency (roughly corresponding to the transfection efficiency) were used for in vivo experiments. Extended data regarding Recombinant DNA and virus perpetration components information can be found in Table S1. crRNA sequences and associated primers can be found in Table S2.

For in vivo sgRNA delivery, pAAV.U6-sgRNA(SapI)-CAG-mRuby2-WPRE-hGHpA was generated by inserting a dsDNA fragment containing the U6 promoter and Streptococcus pyogenes Cas9 guide RNA scaffold with SapI sites for crRNA insertion into MluI-digested pAAV-CAG-mRuby2 ⁷⁰, using NEBuilder HiFi (New England Biolabs, E2621). crRNA sequences were inserted by annealing complementary oligos containing overhangs, followed by ligation into SapI-digested pAAV.U6-sgRNA(SapI)-CAG-mRuby2-WPRE-hGHpA.

AAV production: pAAV.U6-sgRNA-CAG-mRuby2-WPRE-hGHpA containing validated sgRNA sequences was packaged into AAV2-retro ⁷¹ capsids through triple transient transfection of HEK293T cells (ATCC, CRL 3216) with polyethylenimine (Polysciences, 24765-1), followed by cell lysis and purification over iodixanol (Optiprep; Sigma-Millipore, D1556), as previously described ⁷². The AAV2-retro rep-cap plasmid was a gift from Alla Karpova and David Schaffer (Addgene ID: 81070). Viral titers were obtained through qPCR, using a linearized genome plasmid as a standard. Cells were verified to be free of Mycoplasma contamination prior to AAV production.

Fiber photometry recording

FP is a method for measuring population calcium-dependent fluorescence from genetically-defined cell types in deep brain structures using a single optical fiber for both excitation and emission in freely moving mice. A detailed description of the system can be found elsewhere ⁷³. Briefly, our system employed a 490 nm LED for fluorophore excitation (M490F1, Thorlabs; filtered with FF02-472/30-25, Semrock) and a 405 nm LED for isosbestic excitation (M405F1, Thorlabs; filtered with FF01-400/40-25, Semrock), which were modulated at 211 Hz and 531 Hz, respectively. Two systems were used for recording, both controlled by a real-time processor (System 1: RX8-2; System 2: RZ5P, Tucker-Davis Technologies), and delivered to the implanted optical fiber via a 0.48 NA, 400 µm diameter mono-fiber optic patch cable (MFP_400/430/LWMJ-0.48_2 m_FC-ZF1.25, Doric Lenses). The emission signal from isosbestic excitation, which was previously shown to be calcium-independent for GCaMP sensors ^74,75, was used as a reference signal to account for motion artifacts and photobleaching. Emitted light was collected via the patch cable, collimated, filtered after passing through a focusing lens (System 1: MF525-39 filter, Thorlabs, 62–561 focusing lens, Edmunds Optics; System 2: Mini Cube FMC6, Doric Lenses), and detected by a femtowatt photoreceiver (Model 2151, Newport). Photoreceiver signals were demodulated into GCaMP and control (isosbestic) signals, digitized (sampling rates: System 1: 382 Hz; System 2: 6 Hz), and low-pass filtered at 25 Hz using a second-order Butterworth filter with zero phase distortion. A least-squares linear fit was applied to align the 405 nm signal with the 490 nm signal. Then, the fitted 405 nm signal was subtracted from the 490 nm channel and then divided by the fitted 405 nm signal to calculate dF/F values.

Behavioral assays

Estrous Cycle Stage Identification: Estrous cycle stage was recorded following established protocols ⁷⁶. Briefly, vaginal smears (VS) were collected with a 100 or 20 µl pipette holding 15 ml saline at ZT10-14. In cases where the room was entered for injections or light stimulation, VS were taken at the time of interference, to reduce entrainments. VS were flattened and allowed to dry on a microscope slide (Adhesion Superfrost Plus Glass Slides, Brain Research Laboratories) and then stained with cresyl violet dye (0.1%, Sigma-Aldrich, C5042-10G). Smears were then analyzed using light microscopy. Proestrus is characterized by a high number of nucleated epithelial cells. The estrus stage has a high number of cornified epithelial cells. During metestrus there is an increased amount of leukocytes, with the presence of cornified epithelial cells and some nucleated epithelial cells. Diestrus is characterized by mostly leukocytes, with low presence of cornified epithelial cells and nucleated epithelial cells. We defined the number of estrous cycles based on the number of identified proestrous events, even if a full cycle was not detected during the monitored priod. For GCaMP6s data analysis, we categorized each day relatively to the proestrus day; P+0 indicates proestrus day, and P-2, P-1, P+1 and P+2 refer to the days before and after, as indicated. P+1 is always estrus, and P+2 is typically metestrus or estrus. P-2 and P-1 are diestrus or metestrus.

OVX and hormonal replacement: OVX was verified by having a VS profile of diestrus or metestrus. At least four weeks after OVX, sex hormone stimulation was performed as follows; estradiol was administered at 10 μg and progesterone at 500 μg in 0.05 ml of sesame oil delivery s.c. at ZT8, which has been shown to induce sexual receptivity. These estradiol levels are comparable to physiological proestrus peak levels ⁷⁷.

Locomotor activity (LMA) detection: Mice were placed in a rat cage (40 X 34 cm). A PIR motion detector (COMPASS ⁷⁸) was placed 35 cm above the cage bottom, and a Plexiglas cylinder was placed in the middle (10-inch diameter) to ensure even detection of mouse activity. Mice were placed with a tube used for handling to reduce anxiety ⁷⁹.

Chemo- and Optogenetics: After recovery from the surgery, mice were moved to the behavioral room with the light cycle used for the experiment and handled for at least two weeks before the experiment started. Female mice were placed in a rat cage with a round cylinder, as described above. Mice were given at least 3-4 days to acclimate to the new cage setup and handling. Following the first baseline session, the light cycle was changed to DD, with additional light stimulation at CT0 (DD +L0), to preserve their locomotor rhythm as much as possible. At either CT4 or CT10, stimulation was given as follows:

For optogenetics: a 447nm laser (MDL-III-447-200mW, OptoEngine LLC) was used, at 15 Hz, pulse duration of 15 ms, and an intensity of 10 mW, repeated for one-hour total. This pattern was adopted from Mazuski et al (high frequencies pattern) ⁴¹. In addition to the black dental cement, we used two layers of black heat-shrink tubes to prevent interfernces between the relatively high-intensity laser and light-sensitive targets which are not the SCN.

For DREADD: CNO was prepared in distilled water at 1 mg/ml as a stock solution and kept for up to a month at -20 °C. CNO was injected i.p. at 0.1 mg/ml, (0.1 ml per 10 gr) for 1 mg/kg.

To validate opto- and chemogenetic activation post hoc, we activated the cells for one hour under the same conditions or injected CNO one hour before mice were sacrificed (at CT4) and examined c-Fos and ChR2 or hM3Dq expression using IHC.

During all light and neuronal manipulation studies, vaginal smears were taken daily at ~ZT10, except for opto- and chemogenetic experiments, in which they were taken prior to stimulation, to prevent further interference. For these experiments, females were assessed for three weeks for a regular estrous cycle using vaginal smears, and their weight was monitored every three weeks. Females with low number of cycles, <3 over three weeks, were removed from the cohort, but left in the cage for social stability. In one case, a female was excluded from the experiment due to severe weight loss (>10%), and one female was excluded when the estrous cycle was stable throughout all the light manipulations.

Released egg collection: Ovulating eggs were collected from the oviduct the day after ovulation, within 24-30h after the detected proestrus state. Cumulus-oocyte complexes were isolated in M2 media (Sigma, M7167) supplemented with 0.3% hyaluronidase (Sigma, H3506) to determine the number of eggs ovulated.

LiGS 3D histology

LiGS tissue preparation: LiGS histology of implanted mice was performed as described previously ²⁴: after perfusion (20 ml 1× PBS followed by 20 ml 4% PFA), the implant was kept intact; the skin and the lower jaw were gently removed. The remaining skull, including both brain and implant, was placed in 4% PFA for two days. Following fixation, samples were washed in 1 x PBS, then placed in 15% and 30% sucrose solution for cryoprotection. For LED coupling, samples were placed in 22x22 mm disposable embedding molds (70182, EMS) with OCT (Tissue-Tek Compound, Sakura Finetek) and were frozen with an ethanol/dry-ice bath (−78°C). The brain was positioned such that the optical implant was perpendicular to the cube during freezing. Next, a 5 mm LED (Chanzon, yellow) was placed directly above the optical device and secured with additional OCT. To give the brain–OCT cube a flat surface, a 20x40 mm embedding mold (70184, EMS) was filled with OCT while the brain (and coupled LED) was placed upside down and dipped together into the ethanol/dry-ice bath. This process created a large, stable OCT cube that included the sample and the coupled LED. The sample was then cut on one side to expose the LED wires and stored at −80°C until needed.

Light-guided cryo-sectioning: Brains in OCT were placed in a cryostat and sliced from the bottom in 50–100um steps with the LED turned on. To ensure a reproducible light intensity, we used a power supply (DG1022, RIGOL) set to 2.1 V. We used the profile of the light spread by manual observation to define the sectioning endpoint: after the scattered light became sharp, sectioning was continued in small steps (20–50 µm) until a shaded area was seen in the fiber location when the LED was turned off. After sectioning, samples were left at room temperature (RT), allowing the OCT to melt gradually. Samples were then gently placed in a tube and washed with 1× PBS solution.

LiGS staining with IHC: After sectioning, brains were put in 4% PFA for 1–3 hours for additional fixation. The staining protocol was adapted from the iDisco protocol without the pretreatment step [20]. Briefly, the samples were incubated for two days at 37°C in permeabilization solution, followed by two days in blocking solution at 37°C. Next, the samples were incubated with primary antibodies at a 1:200 concentrations for 5–7 days in PTwH/5%DMSO/3%donkey serum, at 37°C. After washing at room temperature (RT) until the next day, samples were incubated with secondary antibody in PTwH/3% donkey serum at 37°C for 5–7 days. Lastly, samples were washed with PTwH at RT until the next day.

LiGS Clearing: Samples were cleared using the SeeDB protocol at RT ⁸⁰.

Thin slice staining:

After perfusion (20 ml 1× PBS followed by 20 ml 4% PFA), brain sections (70 or 100 mm, Leica Vibratome VT1200S) were first incubated in a blocking buffer solution (1× PBS solution with 0.1% Triton X-100 and 10% normal donkey serum) for at least one hour, followed by incubation with primary antibodies in the blocking buffer solution at 4°C overnight. The next day, sections were thoroughly washed four times in 1× PBS (15 min each). Next, the brain sections were transferred to a blocking buffer solution with secondary antibodies and left overnight at 4°C or for two hours at RT. Next, sections were washed as described above and mounted on glass microscope slides (Adhesion Superfrost Plus Glass Slides, Brain Research Laboratories). After the sections were completely dry, they were cover-slipped after applying mounting media (VECTASHIELD® PLUS).

Extended information about IHC materials can be found in Table S1.

Histological imaging

Histological images were obtained with Zeiss 880 confocal microscope at 10X and 40X objectives. Images were analyzed in ImageJ, Matlab, and/or Imaris (Bitplane). Cell identification of GnRH neurons in the MPA was performed with the “Spots” function in Imaris (9.8.0 and 9.9.1), with a diameter of 15 µm, for each channel separately. In addition, the “Statistics” toolbox of Imaris was used to identify overlapped populations (<10 µm). For each animal, three brain slices which included the MPA were quantified, and the injection quality was validated by observing the injection site (Figure S1).

Data analysis

LMA: Following PIR motion detection, the mean activity and onset were calculated using an angular presentation of each 24-hour period, using the “CircStat” toolbox for circular statistics (Matlab ⁸¹). Slopes were calculated using a linear regression function, looking at the mean activity or onset values over days (Matlab, with 95% confidence).

Fiber photometry (FT): dF/F was first aligned using the 405 nm channel, as previously discussed [30]. When z-scored data was presented, we used: dF/F = F- median(F_b)/mad(F) (where ‘mad’ is the median absolute deviation, and F_b is the background signal during the dark phase). The event rate was calculated using the peak finder algorithm (“findpeaks” using “Annotate,” Matlab), with a threshold of 1.2 std above the mean value at the baseline period. For 10 minutes/24h sessions, data was collected starting at ZT8 for 24h. For the analysis, data was shifted by -8 hours. That way, the estrous cycle definition was accurate for the last 8 hours of the light phase, followed by the rest of the light phase and the following dark phase. In the case of proestrus, ovulation occurs during the dark phase, and the following light phase should be considered as estrus. For male-to-female comparison, female data was taken for the proestrus day.

FT FFT analysis: For each dF/F signal (10 minutes/h), FFT was calculated (‘fft’, Matlab). For each hour, integrated FFT power values were calculated for two sets of frequency intervals: a coarse one (a) [0 0.03, 0.03 0.5, 0.5 1] Hz, and a finer one: (b) [0 0.03; 0.03 0.1; 0.1 0.35; 0.35 0.65; 0.65 1.0; 1.0 1.35; 1.35 1.65; 1.65 2.3; 2.3 4] Hz. FFT autocorrelations were calculated for each FFT power interval over the coarse frequency intervals (set (a),‘autocorr’, Matlab). For classification, a spectrogram of the fine frequency intervals was prepared for each session. Each session was tagged by sex and by hormonal state. Prior to machine learning, the spectrogram dimensions were reduced by (1) choosing only frequency ranges between 0 to 1 Hz (the first six intervals), (2) applying PCA (pca, ‘Matlab’), i.e. 24 h x 6 frequencies intervals were reduced from 144 to 12, (3) when indicated, only a subset of the recording 10-minute intervals were used. A leave-one-out cross-validation (LOOCV) approach was used: in each step, one session was left out to be the ‘testing-set’, while the rest of the sessions were used as a ‘training-set’, after randomly choosing the same number of sessions for each state. For training, we used either a “support vector machine” (SVM, ‘fitcecoc’, Matlab), or a “discriminant analysis” (‘fitcdiscr’, Matlab) classifier, followed by prediction (‘predict’ Matlab). The compared states were either male/OVX/P-2 to P+2, or male/OVX/RE/NR. In each classification, two states were compared, averaged over 5 (24h dataset) or 10 cycles (ZT10-13 dataset), and the score was plotted as a matrix, weighted by color (‘matvisual‘ matlab, created by Hristo Zhivomirov). SEM between cycles was 1% on average. (RE- receptive, NR- non-receptive)

Extended information about Software and algorithms can be found in Table S1.

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Data Availability and Code Availability

All data that support the findings of this study are available from the corresponding author on reasonable request.

All codes for the data analysis are available at GradinaruLab/SCN-VIP-estrous-cycle: Anat Kahan, Aug 2022 (github.com)

Acknowledgments:

We would like to thank the Gradinaru lab for helpful discussions; Nikhila Swarna, Shinae Park, and Nathan Appling, for their technical assistance; Prof. Greg Anderson for the GnRH-Ab; Prof. Lance Kriegsfeld for the helpful discussions; and Dr. Catherine Oikonomou for the helpful discussions and manuscript review. This work was funded by the Heritage Medical Research Institute, the Vallee Foundation, and the Center for Molecular and Cellular Neuroscience in the Tianqiao and Chrissy Chen Institute for Neuroscience at Caltech (to V.G.). A.K. was supported by a Caltech Biology and Biological Engineering department postdoctoral fellowship and the Hebrew University Postdoctoral Fellowship for Women, Israel. A.K. and M.B were partially supported by the Global Grand Challenges grant from Bill&Malinda Gates Foundation, G.M.C. was supported by a PGS-D from the National Science and Engineering Research Council (NSERC) of Canada. B.W.B. acknowledges support from the Moore Distinguished Scholar Program at Caltech.

Author contributions

A.K., G.M.C., M.B., and V.G. conceived the study. A.K. G.M.C and M.B. performed the experiments, M.W.B. developed the ML mathematical approach. A.K. analyzed and visualized the data. A.K. and V.G wrote the manuscript. All authors read, reviewed, edited, and approved the final version of the manuscript.

Competing interest declaration

The authors declare no competing financial interests.

There is NO Competing Interest.

FigureS1relatedtoF1GnRHCas9validations.tif
Extended Data 1 Figure S1, related to figure 2: Validation of GnRH-Cre x cas9 injection site and VPAC2 antibody. (A) The injection site, the median eminence (ME). (B) VPAC2 Ab validation based on expression in the SCN.
FigureS2relatetoF4fftanalysis.tif
Extended Data 2 Figure S2, related to Figure 4: FFT spectrogram analysis and additional discriminations of SCN^VIP 24h FP dataset. (A-D) FFT 24h dataset analysis: (A) FFT spectrogram at 0 to 0.03Hz, showing discrimination ability between P-1 and P+1, at ZT9 and 10. The median FFT histogram over samples is shown in dashed lines (B) FFT histogram values at ZT9 vs. ZT10 of P-1 and P+1. Full symbols indicate the center of mass (median). (C) Same as (A), looking at the discrimination ability between OVX and males. (D) FFT histograms at ZT9 vs. ZT10 of OVX and males. Full symbols indicate the center of mass (median). (E) Cross-validated accuracy of SVM classifiers for sex and hormonal state prediction, averaged over five double-sided iterations, using values for ZT7 to 12 only. (F) Cross-validated accuracy with “Discriminate analysis” algorithm to predict receptive state (left) or full estrous cycle days (right). The accuracy values show similar prediction abilities to the SVM algorithm.
FigureS3relatetoF4fftanalysis.tif
Extended Data 3 Figure S3, related to Figure 4: FFT spectrogram creation approach using artificial data. (A) Artificial FP data is a combination of two sine signals, for ZT0-11 and ZT12-23 separately, at frequencies of 0.016 and 1.5Hz, with amplitudes of 10 and 1, as well as 0.02 and 1.0 Hz, with amplitudes of 1 and 0.2, respectively. (B) FP artificial oscillations. (C) Power spectra of the artificial FT data (‘fft’, Matlab), using regular (top) and logarithmic (bottom) scales. Red dots indicate the contributing frequencies, 0.016, 0.02, 1.0, and 1.5 Hz. (D) Integrated FFT over different frequency intervals.
FigureS4relatetoF5DLtransitionv3.tif
Extended Data 4 Figure S4, related to figure 5: FP activity of SCN^VIP neuron at the transition from light to dark (ZT11-13) showing sex differences at event rates, but not at the transition from dark to light (ZT23.5 to 0.5). (A) SCN^VIP FP activity at ZT11-13 (i) Representative examples of FP recordings from a female (red) and male (blue) with peak identification (blue triangles). (ii) Averaged responses (mean±sem, sem in gray. females: n=14, males: n=5, averaged for at least three repeats each). (iii-iv) Mean dF/F and event rates, compared between males and females at ZT 11-12 and ZT 12-13 (mean±sem, with individual values shown). Event rates were higher during the light phase from ZT 11-12 compared to ZT 12-13 in females but not in males (0.58±0.06 and 0.64±0.05 events/min at ZT11-12 vs. 0.31±0.04 and 0.32±0.04 at ZT12-13, There was no significant difference in mean dF/F (3.1±0.1 and 2.69±0.08 a.u. at ZT11-12 vs. 2.9±0.2 and 2.47±0.09 a.u. at ZT12-13, males and females respectively. (B) SCN^VIP FP activity at ZT23.5-0.5. (i) Averaged responses (mean±sem, sem in gray, females: n = 7, males: n = 5, averaged for at least three repeats each). (ii) Mean dF/F, showing no differences between males and females and a significant increase from dark to light. (iii) Event rates show no differences between males and females and significantly increase from dark to light. Nonparametric Kruskal-Wallis test, * p <0.05.
FigureS5relatetoF5fftanalysis.tif
Extended Data 5 Figure S5, related to figure 5. Activity, FFT spectrogram, and discriminations analysis of ZT10-13 FP data. (A) Averaged event rates (left) dF/F (right) at ZT10-13 across estrous states. (B) An example of FFT spectrogram of an individual female, across estrous days, followed by OVX and hormonal induction, average over at least three repeats. (C) Cross-validated accuracy of SVM classifiers for discriminating at ZT11-12 can produce above chance classification between P-1 and P+0 (Proestrus) P+1 (Estrus), but not between P-2 and the rest of the states, as was found for ZT10-10.5. (D) Cross-validated accuracy of “Discriminate analysis” algorithm with FFT spectrogram at ZT10-10.5, compared to SMA (Figure 5). Accuracy values are very similar.
FigureS6relatetoF6chemo.tif
Extended Data 6 Figure S6, related to Figure 6. Time-restricted SCN^VIP neuron activation with excitatory DREADD in the afternoon is insufficient to rescue estrous cycle irregularity. (A) The experimental design. VIP-Cre females were injected with Gq expressing virus (Exp, n=6) or eYFP (Ctrl, n=4), and put under four conditions, LD, DD+L0, DD+L0+C4, and DD+L0+C10 (L0, C4, and C10 correspond to light at CT0, CNO at CT4 and CT10; L0: light on for 0.5h; C: CNO application (1mg/kg, cyan). (B) The number of estrous cycles in three weeks, under the three conditions shown in A. (C) Post hoc histology, showing virus expression, mCherry (orange, Exp) and eYFP (green, Ctrl), with c-Fos staining (purple), ~1h after CNO application, absent from the Ctrl group.
FigureS7relatetoF6lightmanipulationLMA.tif
Extended Data 7 Figure S7, related to figure 6. LMA under different light conditions of VIP-cre female mice. (A) LMA under each light condition of one of the cages. (B) Daily onset (left) averaged (middle) LAM (n cages= 2), presented over days. Each cage had three females and a running wheel for enrichment.
FigureS8relatetoF6opto.tif
Extended Data 8 Figure S8, related to figure 6: SCN^VIP optogenetic activation in the late afternoon is insufficient to rescue estrous cycle regularity. (A-B) Time-restricted SCN^VIP neurons activation with ChR2. (A) The experimental design. VIP-Cre females crossed to ChR2 (ctrl: n=5, 447 nm excitation: n=8) or GFP reporter lines (n=3), put under three conditions, LD, DD+L0+O4 (447nm excitation at CT4) and DD+L0+O10 (447 nm excitation at CT10, blue, L0: light on at CT0 for 0.5h). (B) The number of estrous cycles in three weeks, under the three conditions shown in D. (C) Detailed LiGS histology of SCN^VIP neurons below the optical implant. Examples of LiGS histology for post hoc fiber location verification and c-Fos expression. (i) VIP-Cre x ChR2 mouse with no neuronal activation. (ii) VIP-Cre x ChR2 mouse with neuronal activation, showing c-Fos expression ~1h after illumination with 447 nm laser, at 3D (top) and 2D (bottom). (iii) VIP-Cre x GFP mouse with c-Fos. Illustration of the fiber in gray, ChR2 or GFP signal, indicative of VIP neurons in green, c-Fos staining ~1h after illumination with 447 nm laser (magenta), showing no expression. 447nm laser illumination was done around ZT 13 for 1h, the same conditions as during experiments.
EDTable1REAGENTorRESOURCE.docx
Extended Data 9 Table S1: Detailed reagent and resources.
EDTable2sgRNA.docx
Extended Data 10 Table S2: related to figure 2. crRNA sequences and associated primers for Genetic knockout in GnRH neurons using CRISPR

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Estrous Cycle and Egg Release Resets with Light Entrainment Inspired by Neurodynamics in the Circadian Pacemaker

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