82675015 - 2023.01.14.524075v1.full.pdf

Dysregulated mammalian estrus cycle rescued by timed activation

of VIP

neurons in the circadian pacem

aker and late afternoon light exposure

Estrus cycle rescued by afternoon activation of suprachiasmatic

VIP neurons

One Sentence Summary:

Modulating and recording the activity of suprachiasmatic VIP ne

urons in freely

behaving mice reveals their regulation of fertility by mediatin

g the response to late afternoon light.

Anat Kahan

, Gerard M. Coughlin

, Máté Borsos

, Bingni W. Brunton

, Viviana Gradinaru

Division of Biology and Biological Engineering, California Inst

itute of Technology, Pasadena,

California, 91125, USA

Department of Biology, University of Washington, Seattle, Washi

ngton, 98195, USA

* Correspondence to V.G. (

viviana@caltech.edu

) and A.K. (

anatk@caltech.edu

)

Abstract

Jet lag and shift work disrupt the menstrual cycle and decrease

fertility. The circadian pacemaker, the

suprachiasmatic nucleus (SCN),

is known to modulate ovulation,

but the mechanism is unclear. Here we

explore this connection by tracking the dynamics of vasoactive

intestinal peptide (VIP)-expressing neurons

in the SCN in freely-behavi

ng mice. We show that SCN

VIP

activity is time-of-day-

and sex-dependent, and

estrous-state-dependent in late

afternoon, gating downstream ac

tivation of GnRH neurons. Afternoon light,

as well as specific activation of SCN

VIP

neurons, rescues estrous cycle regularity and egg release in a

nimals

in altered light conditions, emphasizing the role of SCN

VIP

neurons as a time-dependent light-responsive

switch. Our results reveal the dynamic mechanism by which SCN

VIP

neurons mediate light responses to

regulate estrous states and demonstrate light-induced fertility

rescue.

Keywords

: Estrous cycle; suprachiasmatic

nucleus; vasoactive intestinal

peptide; circadian rhythm;

GnRH; calcium imaging; fiber photometry; CRISPR

Main

In the mammalian estrous cycle,

fluctuations in sex hormone sec

retion occur with periods longer than a

day, and these infradian rhythms are coordinated with the behavioral circadian rhythm to align the fertility

window with social interaction and receptivity, thus maximizing

reproduction (

). The critical event of

ovulation is controlled by the h

ypothalamic-pituitary-gonadal (

HPG) axis and, in mice, is synchronized

with the dark phase every four or five days. In metestrus and d

iestrus, estrogen secretion from the ovary

increases, followed by a luteinizing hormone (LH) surge in the

late afternoon of proestrus, triggering

ovulation ~12 hours later. Hypotha

lamic neuronal populations su

ch as gonadotropin-releasing hormone

(GnRH) neurons in the medial preoptic area (MPA) (

), RF-amide-related peptide-3 (RFRP-3) neurons in

the dorsomedial hypothalamus (DMH) (

) and kisspeptin (KISS1) neurons in the anteroventral

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periventricular nucleus (AVPV) (

4, 5

) and the arcuate nucleus (

) mediate estrogen feedback and are

responsible for the LH surge.

The hypothalamic suprachiasmatic

nucleus (SCN), the circadian r

hythm pacemaker, is fundamental to

ovulation occurrence. Several pieces of evidence suggest that s

ignals from the SCN directly generate the

LH surge. First, the SCN has ne

uronal projections to relevant h

ypothalamic regions, including the MPA

(

), AVPV (

), 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 (

), and neurons expressing these peptides project directly or

indirectly to GnRH (

16-18

), Kiss1 (

), and RFRP-3 (

) 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 a

pposition to GnRH neurons (

7, 19

). Based on bilateral differences in c-Fos

expression in GnRH neurons in hamsters, it was suggested that c

ircadian regulation of GnRH neurons is

mediated by direct synaptic input to GnRH neurons (

). While VIP is known to excite GnRH neurons in

brain slices, the estrous cycle regulation of this process is d

ebated in the literature (

16, 18, 21

), and the

infradian dynamics of VIP-expr

essing neurons throughout the est

rous cycle are unknown.

Here, we show that light information controls estrous cycle regularity through SCN

VIP

neurons. Our

in vivo

approach combines neuronal and physiological outcomes, which ca

n be tracked long-term. Measuring the

dynamics of SCN

VIP

neurons across the estrous cycle using a calcium indicator wit

h fiber photometry

followed by advanced machine-learning-assisted signal analysis,

we show that SCN

VIP

neuronal activity is

sex-specific and light-active, with increased activity in the l

ate afternoon, especially when females are non-

receptive. We observed a reduction in estrous cycle regularity

when VPAC2 was downregulated in GnRH

neurons, suggesting that this time-of-day information is gating

the estrous cycle through GnRH neurons.

Based on these observations, we hypothesized that afternoon lig

ht could rescue estrous cycle regularity

under minimal dark conditions, as indeed we found, together wit

h the rescue of egg release. Furthermore,

this rescue could be mimicked by specific SCN

VIP

activation in the afternoon, emphasizing the unique role

of SCN

VIP

neurons as a light switch. Together, this work establishes a s

olid mechanistic connection between

light, SCN

VIP

activity, estrous cycle, and fertility.

Results

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., d

uring jet lag) are associated with irregular

menstrual cycling and decreased fertility. To confirm the signi

ficance of light regularity on the mouse

estrous cycle, we performed a "jet lag" experiment in which we

housed female C57 mice in 12:12 hour

light:dark (LD) conditions, and every 4-5 days, advanced the li

ght in the room by six hours. This pattern

was previously found to dramatically decrease pregnancy success

in mice (

), and reduce general fertility

in humans (

23, 24

). Using cytology of daily vaginal smears over three weeks (4-5

cycles in non-jet-lagged

animals), we found that this light pattern decreased the regula

rity of the estrous cycle in the mice, reducing

the number of proestrus events from 4.6±0.2 to 2.1±0.3 (mean±se

m) in three weeks. This effect was due

mainly to increased time spent in

estrus, from 7.0±0.6 to 9.1±0

.9 days (Figure 1A-D).

The SCN is one of the initial brain relays for retinal response

s to light, and SCN

VIP

neurons were previously

identified as essential to the circadian response to light (

25-29

), forming monosynaptic connections to

intrinsically photosensitive retinal ganglion cells (ipRGCs) (

27, 30

) and mediating the rapid response to

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light during the dark phase (

). We, therefore, tested the effect of specific ablation of SCN

VIP

neurons on

estrous cycle regularity in female mice kept under 12:12 condi

tions. We injected adeno-associated virus

(AAV) to deliver Cre-dependent caspase-3 to the SCN of VIP-Cre

mice,

inducing targeted

apoptosis of

SCN

VIP

neurons (Figure 1E). We found that ablation significantly reduced the number of proestrus

repetitions, from 4.3±0.4 to 2.8±0.3 in three weeks, significantly lower than the control group (Figure 1F-

H). These results support a specific role for SCN

VIP

neurons in connecting light stimulus with estrous cycle

regulation.

SCN

VIP

activity is sex-specific and time-of-day-dependent

To characterize the temporal dynamics of SCN

VIP

neuron activity,

we used fiber photometry (FP) to record

bulk neuronal activity in males and females using a genetically

encoded calcium indicator, GCaMP6s, using

VIP-Cre x GCaMP6s mice, implanted with two 400



m optical fibers. To verify the fiber location, we used

light-guided-sectioning (LiGS) (

), a 3D histological method that we developed to determine the

precise

position of optical implants within deep and small brain nuclei

, such as the SCN (Figure 2A). First, we

tested the responsiveness of the calcium signal to the light phase transitions, zeitgeber time (ZT) 11 to 13

and ZT23.5 to 0.5. Using dF/F and event-rate analyses, we found

that SCN

VIP

activity is synchronized with

the light change (Figure S1). Having thus confirmed light sensi

tivity at the light phase transitions, we

recorded bulk GCaMP6s activity for 10 minutes each hour over 24

hours (Figure 2B). We determined the

estrous stage of cycling females by vaginal smear cytology over

multiple continuous days until at least three

100

cycles were recorded, and compared their SCN

VIP

activity to that of males and ovariectomized (OVX)

101

females, recorded for at least three days each. All animals sho

wed circadian fluctuations, with reduced

102

SCN

VIP

activity during the dark hours of ZT12-24, based on both event rates (Figure 2C) and dF/F (Figure

103

S2A-B). Females had higher integrated dF/F than males during the light phase (8.7±0.8 and 3.5±0.5 a.u.,

104

females and males, respectively, Figure S2A-C), but we did not identify significant differences in bulk

105

activity between estrous stages or between intact and ovariecto

mized females (Figure 2D-E). We observed

106

significantly increased event rates during the light phase for

all estrous stages (0.36±0.04 vs. 0±0

107

events/minute, light vs. dark, averaged over estrous stages) an

d increased dF/F values for some (Figure

108

S2E). Interestingly, we noticed that both event rate and dF/F e

xhibited parabolic activity during the light

109

phase (Figure 2E and Figure S2F, averaged R

=0.8±0.07 mean±sem, ZT2 to 11), decreasing to an average

110

minimum at ZT5 and then increasing again to a maximum at ZT11.

By comparing specific time windows,

111

we observed a trend of higher event rates in M/D compared to E

during the late afternoon (ZT11-12), but

112

none of the comparisons showed a significant difference between

females in different estrous stages or

113

OVX (Figure S2G). Based on this analysis, FP data suggest that

the main role of SCN

VIP

neuronal activity,

114

which is sex-dependent, is a permissive circadian switch aligne

d with light information to convey the best

115

timing to ensure fertility.

116

FP is a bulk measurement. Reasoning that subpopulations of VIP

neurons within the SCN may have

117

different activity properties (

), we applied fast Fourier transform (FFT) analysis, a method to transform

118

time-series data from the time to the frequency domain, and the

refore separate the frequencies of time-

119

variant oscillators (

). We applied FFT to each 10 minutes recording-period, for each

24h session and

120

identified seven frequency ranges (from 0.003 to 1.35 Hz) with

distinct log-log profiles (Figure 2F).

121

Integrating the power spectra over these frequency ranges showe

d light-dark rhythmicity (Figure 2G), and

122

autocorrelation over the multiple days of recording revealed ci

rcadian phase in all frequency ranges (Figure

123

2H), with low amplitude at the lowest range (0.0033 to 0.007 Hz

), which therefore was not included in the

124

following analyses. Averaging the integrated power spectra of i

ndividual animals across experimental

125

groups showed a clear difference

in the amplitutes of the low-f

requency SCN

VIP

contributions between

126

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males and females (Fi

gure 2I, note the diffe

rent color bar range), all showing clear circadian profile, time-

127

of-day dependent.

128

129

Late-afternoon SCN

VIP

activity correlates with estrous state

130

Next, we aim to quantify the differences in the FFT spectrogram

s, focusing on the uniqueness of the late

131

afternoon, which is the time frame of the LH surge. We, therefo

re, looked at differences in low-frequency

132

FFT signal between estrous states. FFT histograms of males was

distinct from OVX females throughout

133

the light period (Figure 3A). By contrast, we saw a time-of-day specific difference in FFT histograms

134

between cycling females on non-receptive (M/D) and receptive (E

) days, with a separation at ZT9, but not

135

ZT4 (Figure 3B). To further investigate this critical window, w

e tested whether the qualitative differences

136

in FFT histograms we observed were sufficient for a machine-lea

rning-based classifier to discriminate

137

between states. We applied a l

eave-one-out cross-validation (LOOCV) approach to the FFT spectrograms,

138

including frequencies up to 1 Hz, as extracted from individual

10 minutes/hour FP daily trials of males,

139

OVX females, and cycling females. In each round, an even number

of trials from two conditions was used

140

to train the classifier, which was then tested on an additional

trial not included in the training set (using

141

“Support vector machine” algorithm, SVM, see Methods for detail

s). The overall score reflects the

142

prediction success of the classifier over all rounds. As expected, the classifier was able to distinguish

143

between males and females, with a

n average accuracy score of 74

%. Interestingly, the classifier could also

144

distinguish OVX females from both males and other females (Figu

re 3C), reflecting the increased

145

sensitivity of the FFT approach com

pared to simple event detection.

146

We used the cross-validation score to test whether within the t

otal 24h of activity there is a relevant time

147

period for estrous cycle classification, focusing on the abilit

y to distinguish between before ovulation (M/D)

148

and immediately after ovulation (E). Indeed, the prediction ability between M/D and E increased at the late

149

afternoon with a three hours time window, which increased the c

hance level at ZT7-10, with maximal

150

discrimination ability at ZT9-11 (Figure 3D). Using only this c

ritical window of ZT 9-11 enabled the

151

classifier to predict females’ receptivity status, E vs. D/M, w

ith an accuracy of 74% (Figure 3F, supported

152

by using an alternative algorithm, Figure S4), and allows above

change classification between before and

153

after ovulation (M/D vs. P).

154

Reasoning that we might have missed transient changes in SCN

VIP

signal in the critical window by recording

155

only 10 minutes per hour, we repeated our FP experiment with co

ntinuous recording from ZT 10-10.5, the

156

approximate time of the LH surge during proestrus, and from ZT

11-13. We observed a significant decrease

157

in event rate at ZT10 when females were in E, compared to M/D (

Figure 3H), in line with the trend we

158

observed with 10 minutes per hour sampling (Figure 2D). We next applied FFT analysis, binning the

159

continuous recordings into ten-minute intervals (Figure 3I), an

d found that the classifier could now

160

discriminate not only between females in M/D and E (59.8±0.3%,

mean±sem, over ten iterations) but also

161

between OVX females and females in P or E, with prediction succ

ess over 65% (Figure 3J).

162

Since the SCN is estrogen-sensitive (

33-35

), we decided to test whether hormonal stimulation of proestrus

163

in OVX females would recover a receptive pattern of SCN

VIP

activity. However, hormonal induction with

164

estrogen (OVX+E) followed by progesterone (OVX+E+P) did not sig

nificantly alter SCN

VIP

activity at

165

ZT10 (Figure S5B), suggesting that the ovariectomy had already

caused irreversible changes in the SCN

166

(

). Together, our FP results show a late afternoon decrease in S

VIP

activity during the receptive period,

167

possibly indicating a mechanism to inhibit the downstream HPG t

arget just before and after ovulation.

168

Loss of VIP receptors on GnRH neurons disrupts estrous cycle regularity

169

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Our data suggest that SCN

VIP

neurons act as a permissive circadian switch, integrating ligh

t information to

170

inform optimal fertility timing. We next sought to identify the

target of this switch. Prior slice

171

electrophysiology showed that VIP increases the activity of GnR

H neurons in a time-of-day dependent

172

manner (

16, 18

). While these results are suggestive of a direct effect, it could also be indirect, affecting

173

local SCN interactions, such as to AVP neurons, which further p

roject to Kiss1 neurons (

36, 37

), the pulse

174

generator for GnRH neurons (

), or via ovarian circadian control (

12, 39

). To test whether SCN

VIP

neurons

175

transmit light information directly to GnRH neurons, we used Clustered Regularly

Interspaced Short

176

Palindromic

Repeats

(

CRISPR) genetic editing to disrupt either VPAC2 or Kiss1 recept

or (KISS1R)

177

expression specifically in MPA

GnRH

neurons that project to the median eminence (ME) by AAVretro

178

injection into the ME (

) (Figure 4A). We verified MPA

GnRH

transduction and receptor knockdown by

179

immunostaining (Figure 4B-D), which showed a significant physio

logical effect despite low transduction

180

efficiency. Female mice were kept in 12:12 LD conditions, and t

heir estrous cycles tracked for three weeks

181

prior to and after CRISPR editing (following a two-week recovery period after surgery). While the control

182

group exhibited a regular estr

ous cycle, with an average freque

ncy of five proestrus events in three weeks,

183

knockdown of either

kiss1r

vipr2

resulted in a significant reduc

tion in proestrus events (from

4.2±0.3 to

184

2.7±0.3, p=0.0 for

kiss1r

and from 4.8±0.3 to 2.8±0.3, p=0.007, for

vipr2

, Figure 4E). Similar to what we

185

observed with SCN

VIP

ablation, females with disrupted VPAC2 expression also spent s

ignificantly more

186

time in estrus (Figure 4F). These results suggest that SCN

VIP

act directly on GnRH neurons through VIP

187

release, to further regulate the estrous cycle.

188

Afternoon light rescues estrous cycle regularity

and egg release under limited light conditions

189

The time-of-day-dependent activity of SCN

VIP

neurons that we observed inspired us to test whether light

190

exposure in the afternoon sensitiv

e time window w

ould be suffic

ient to rescue circadian-disrupted fertility

191

in vivo

. To limit light exposur

e while minimally affecting locomotor activity, we group-housed females in

192

constant-dark conditions (DD) with a 30-minute pulse of light a

t circadian time (CT) 0 (DD+CT0

0.5L

)

193

(Figure S7). We then tested different light stimulus regimes se

quentially for three weeks each while tracking

194

estrous stages: LD, DD, DD+CT0

0.5L

, and two conditions with an add

itional hour of light, the firs

t at CT10

195

(DD+CT0

0.5L

+CT10

) and the last at CT4 (DD+CT0

0.5L

+CT4

Figure 5A). During the LD period, we

196

observed an average of 4.2±0.5 proestrus events in three weeks,

indicative of a normal estrous cycle. DD

197

and DD+CT0

0.5L

conditions reduced this to 2.8±0.8 and 1.8±0.3 proestrus event

s in three weeks,

198

respectively. Notably, one hour of light at CT10, but not CT4,

was sufficient to rescue

the regularity of the

199

estrous cycle, with 4.8±0.3 proest

rus events in three weeks and

regular distribution of estrous states (Figure

200

5B-C). To directly assay fertility outcome, we also measured th

e number of eggs released to the oviduct on

201

the day following ovulation, identified by the transition from

proestrus to estrus. We avoided using

202

pregnancy success as a fertility readout since pregnancy itself

might involve circadian clock cues (

203

Compared to age-matched controls in 12:12 LD conditions, female

s in DD+CT0

0.5L

+CT10

conditions

204

released approximately twice as many eggs as those in DD+CT0

0.5L

conditions (Figure 5D). Together, these

205

results show that ambient light in the late afternoon is critic

al for both estrous cycle regularity and increased

206

egg release.

207

Activation of SCN

VIP

neurons in the afternoon rescues estrous cycle regularity

208

Based on the ability of afternoon light to rescue estrous cycle

regulation under limite

d light conditions, we

209

hypothesized that specific SCN

VIP

activation could do the same. W

e first tried an optogenetic ap

proach in

210

VIP-Cre x ChR2 mice using a similar temporal activation paradigm (Figure S9A). ChR2 activation at CT10

211

rescued proestrus frequency in half of the experimental cohort

(VIP-Cre x ChR2, 4 females out of 8),

212

compared to one VIP-Cre x ChR2 animal in the control group (no

excitation) (Figure S9B). While we

213

verified fiber localization using

post hoc

LiGS histology and activation of SCN

VIP

neurons by c-Fos labeling

214

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under the fiber (Figure S9C), it is possible that the variable

results were due to differences in fiber

215

localization affecting the distribution of the light relative t

o the core of the SCN, heterogeneous distribution

216

of neuronal subpopulations, or asymmetric connectivity of the S

CN to the ovary (

217

While both optogenetic and chemogenetic methods have been used

previously for SCN

VIP

neurons (

26, 31,

218

42, 43

chemogenetic approaches produce longer time scales of activatio

n, which might better represent

219

the natural activation of SCN

VIP

neurons. We therefore used a single injection of clozapine N-o

xide (CNO),

220

a synthetic drug, to induce calcium flux through an excitatory

Designer Receptor Exclusively Activated by

221

Designer Drugs (DREADD), hM3Dq, delivered to the SCN by AAV injection (Figure 5E). We verified

222

CNO activation

post hoc

by c-Fos expression (Figure 5F). Again, we tested the estrous

cycle regularity of

223

experimental and control (inject

ed with AAV not carrying hM3Dq)

groups over four sequential conditions

224

(three weeks each): LD, DD+CT0

0.5L

, and DD+CT0

0.5L

with CNO injection at either CT4 or CT10 (Figure

225

5E). We found that activation of SCN

VIP

neurons in the critical time window at CT10, but not earlier i

n the

226

day, rescued the regularity of the estrous cycle (Figure 5G-I).

227

Discussion

228

Here we novelty show that either jet lag or specific ablation o

f SCN

VIP

neurons significantly reduces estrous

229

cycle regularity in mice. By measuring bulk calcium dynamics of

SCN

VIP

neurons over sequential days, we

230

found that SCN

VIP

neurons are primarily light-ent

rained and that their activity

levels cycle over the light

231

period. Using FFT analysis and advanced machine learning (ML)-b

ased discrimination, we identified

232

significant differences in activit

y patterns between males, nat

urally cycling, and ovariectomized females.

233

In addition, we identified a difference in SCN

VIP

activity at the peak time of ZT10 during receptive days.

234

While ML has been used to identify sex diffe

rences in biologica

l samples or behaviors (

44-47

) and estrous

235

cycle stages from vaginal smears (

48-50

), this is, to our knowledge, the first demonstration of its us

e to

236

identify estrous cycle states based on neuronal activity. Hypot

hesizing that SCN

VIP

activity at ZT10 acts as

237

a permissive signal to convey th

e best timing to ensure fertili

ty, we found that SCN

VIP

excitation either

238

through the retinohypothalamic tract via light or via chemogene

tically-specific activation at this unique

239

feedback timepoint of ZT10 rescued estrous cycle regularity. To

our surprise, afternoon light also rescued

240

the number of eggs released in limited-light conditions, indica

ting a significant improvement in fertility.

241

While the collection of released eggs is not commonly performed in naturally cycling mice, we were able

242

to do so due to our close monitoring of the estrous cycle, thereby avoiding confounding effects of the

243

exogenous hormones required to induce superovulation (

244

While SCN

VIP

neuronal activity is known to participate in the control of the HPG axis, here we show for

245

the first time, to our knowledge, that it is sex and ovarian-de

pendent

in vivo

. In the future, it will be of

246

interest to determine the mechanism of hormonal control. In add

ition to a previously-reported dependency

247

on estrogen (

25, 33-35

), we suspect progesterone is also involved, based on hypothala

mic single-cell

248

RNAseq data suggesting that

VIP-targeted

neurons co-express progesterone receptor (PR) mRNA (

249

While we attempted to test this dependency

in vivo

using OVX females, we did not observe a recovery of

250

receptive-like activity in hormonal

stimulation paradigms; the

long period following ovariectomy without

251

hormonal replacement may have caused a loss of plasticity that

brief hormonal treatment could not rescue.

252

The decreased ovulation we observed in a minimal light schedule

aligns with previous reports of reduced

253

estrous cycle regularity in response to altered LD (

) and DD schedules (

54, 55

). A role for VIP was first

254

established by showing that a full-body VIP-KO female mouse had reduced estrous cycle regularity (

)

255

and previous studies have estab

lished VIP-GnRH interaction, sho

wing that VIP excites GnRH neurons in

256

brain slices (

16, 18

) and reporting anatomical connectivity (

7, 17, 19

). Here, using new technologies,

257

including opto- and chemogenetics, CRISPR gene editing and vira

l delivery, we were able to pinpoint the

258

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physiological effect on fertility to VIP neurons located in the

SCN and downstream VIP receptors on GnRH

259

neurons in the MPA. Note that other VIPergic populations outsid

e the SCN might also signal to GnRH

260

neurons through VPAC2 (

). In addition, VIP could also act through other paths, includi

ng local

261

suprachiasmatic stimulation of AVP neurons, which are known to

regulate Kiss1 neurons, or via peripheral

262

stimulation of the ovary (

12, 41

). Consistent with the former, we observed estrous cycle disrup

tion by

263

knockdown of KISS1R in MPA

GnRH

neurons. We also note that SCN

VIP

neurons are not homogenous in at

264

least two aspects. First, SCN

VIP

neurons release two neuropeptides, VIP and GABA (

). We examined the

265

role of VIP because it was shown that the calcium activity of S

VIP

neurons correlates with VIP release

266

(

), but we cannot rule out an additional role for GABA. Second,

recent studies have functionally (

) and

267

genetically (

27, 58

) defined two subpopulations of SCN

VIP

neurons. It will be interesting to address these

268

aspects in the future.

269

Based on our results, we suggest the following model: circadian

oscillation of SCN

VIP

neuronal activity

270

gives a late-afternoon permissive input, with increased event rate during metestrus and diestrus, to

271

downstream targets such as MPA

GnRH

neurons, mediated by direct VIP signaling to GnRH neurons, as

well

272

as to other hypothalamic ce

ll populations,

such as SCN

AVP

and possibly to the ovaries. SCN

AVP

neurons in

273

turn, signal to Kiss1 neurons, which, together with the direct

SCN

VIP

signal, ensure increased GnRH activity

274

to drive the LH surge during proestrous. Following the LH surge

, estrogen levels drop and progesterone

275

levels rise, causing a decrease in SCN

VIP

neuronal activity in this critical window of the following day

276

(estrus). We suggest that this reduction in activity functions

to prevent the reoccurrence of ovulation,

277

possibly via reduced stimulati

on of GnRH neurons and increased

activity of DMH

RFRP-3

neurons (

278

Our work here in mice offers a model of a combination between c

ircuitry and dynamics for studying the

279

HPG axis and its neuronal control by the circadian rhythm pacem

aker. This approach may be particularly

280

useful in an era of circadian disruption due to artificial ligh

t and with increasing evidence that shifted

281

working hours are associated with human reproductive disorders

(

). In particular, our observation of

282

increased egg release in response to targeted light intervention may inspire potential interventions for

283

women who struggle with infertility due to shifted working hour

s or frequent jet lag (

60-62

284

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;

https://doi.org/10.1101/2023.01.14.524075

doi:

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Data Availability and Code Availability

441

All data that support the findi

ngs of this study are available

from the corresponding author on reasonable

442

request.

443

All codes for the data analysis are available at

444

GradinaruLab/SCN‐VIP‐estrous‐c

ycle: Anat Kahan

, Jan 2023 (githu

b.com)

445

446

447

Acknowledgments

448

We would like to thank the Gradinaru lab for helpful discussion

s; Nikhila Swarna, Shinae Park, and Nathan

449

Appling, for their technical assistance; Prof. Greg Anderson fo

r the GnRH-Ab; Prof. Lance Kriegsfeld for

450

the helpful discussions; and Dr. Catherine Oikonomou for the he

lpful discussions and manuscript review.

451

This work was funded by the Heritage Medical Research Institute

, the Vallee Foundation, and the Center

452

for Molecular and Cellular Neuroscience in the Tianqiao and Chr

issy Chen Institute for Neuroscience at

453

Caltech (to V.G.). A.K. was supported by a Caltech Biology and Biological Engineering divisional

454

postdoctoral fellowship and the

Hebrew University Postdoctoral

Fellowship for Women, Israel. A.K.

455

acknowledges the support of her husband and children. A.K. and

M.B were partially supported by the

456

Global Grand Challenges grant from Bill & Melinda Gates Foundat

ion. G.M.C. was supported by a PGS-

457

D scholarship from the National Science and Engineering Researc

h Council (NSERC) of Canada. B.W.B.

458

acknowledges support from the Moore Distinguished Scholar Progr

am at Caltech.

459

Author contributions

460

A.K., G.M.C., M.B., and V.G. conceived the study. A.K., G.M.C.,

and M.B. performed the experiments,

461

B.W.B. and A.K. developed the ML mathematical approach. A.K. an

alyzed and visualized the data. A.K.

462

wrote the first draft of the manuscript. All authors read, revi

ewed, edited, and approved the final version of

463

the manuscript.

464

465

Competing interest declaration

466

The authors declare no competing financial interests.

467

468

469

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;

https://doi.org/10.1101/2023.01.14.524075

doi:

bioRxiv preprint

Figures and Figure legends

470

471

Figure 1: Estrous cycle regularity is significantly reduced by

either jet lag or specific ablation of

472

SCN

VIP

neurons.

(A) Jet lag experimental design, with light advanced shifts eq

uivalent to flying from New

473

York to Honolulu, four days later to Hong Kong, followed by Zur

ich and back to New York, all in three

474

weeks. The gray asterisk

indicates the time of vaginal smear collection. (B) Examples of

vaginal smear

475

traces of two control (top) vs. two jet-lagged (bottom) mice. P

- proestrus, E-estrus, M- metestrus, D-

476

Diestrus. (C) Distribution of estrous-cycle stages in C57 mouse

females in jet-lagged vs. control periods

477

(Ctrl: n=7, Jet lag: n=7). (D) Boxplot showing the number of da

ys spent in each estrous stage in a three-

478

week. (E) SCN

VIP

ablation viral approach and VIP antibody staining in the SCN o

f a representative

479

experiment. Experimental (CASP3, n=6) and control (Ctrl, n=6). (F) Female mice were placed under 12:12

480

LD conditions, and VS were collected in the late afternoon (gra

y asterisk) for three weeks, before and after

481

virus injection, with two weeks

of recovery after the surgery.

(G) Examples of vaginal smear traces of two

482

control (top) vs. two CASP3 injected (bottom) mice. The gray li

ne separates before and after injection. (H)

483

The number of proestrus events in three weeks, before and after

injection of Cre-dependent CASP3 or

484

control (Ctrl) virus. (I) Boxplot showing the number of days sp

ent in each estrous stage in a three-week,

485

before and after virus injecti

on (*p<0.05, ***p<0.005; Nonparam

etric Kruskal–Wallis test. Two sample t-

486

test when data was compared w

ithin two conditions and was normally distributed).

487

488

Figure 2: Temporal changes in SCN

VIP

activity show circadian patterns of event rate and FFT

489

spectrogram.

(A) The experimental setup: VIP-Cre crossed to Ai162-reporter l

ine to express GCaMP6s.

490

The fiber location is validated with light-guided-sectioning (L

iGS) histology (

). Mice were kept under

491

12:12 LD conditions. VS were taken in the late afternoon (gray

asterisk) (B) A representative example of

492

10-minute-per-hour recording, over 24 hours, from one female. (

C) Event rates over 24h

of recording across

493

estrous states (n females=8, n OVX=5, each state is represented

at least three times in each female). (D)

494

Event rates averaged over dark and light phases. For clarity, s

ignificance is marked only between adjacent

495

periods. Nonparametric Kruskal-Wallis test, followed by Tukey's

correction (D) (*p<0.05; **<0.01). (E)

496

Parabolic fits to event rates during ZT2-11, showing minimum at ZT5 and maximum at ZT11. (F-J) FFT

497

analysis (See Figure S3 and Methods for details). (F) FFT power

vs. frequency for one session of one animal

498

(logarithmic scale). Each line represents 10 minutes of recordi

ng during the dark phase (black) and the light

499

phase (yellow). The seven gray li

nes represent the integrated frequency intervals: 0.003 to 0.007, 0.007 to

500

0.05, 0.05 to 0.1, 0.1 to 0.25, 0.25 to 0.45, 0.45 to 1.0, and

1.0 to 1.35 Hz, respectively. (G) Integrated FFT

501

powers of the frequency interval

s shown in (F) over 24h, showin

g one cycle of light-dark response. (H)

502

FFT autocorrelation over days for four of the frequency interva

ls, showing circadian modulations. The

503

interval of 0.003 to 0.007 Hz has reduced circadian phenotype a

nd therefore removed from further analysis.

504

(I) Averaged FFT spectrograms for all recorded sessions by sex

and hormonal state, showing clear 12:12

505

LD contributions.

506

507

Figure 3: Estrous-cycle is discriminated in late afternoon with

FFT classification and additional wide

508

temporal window afternoon recording of SCN

VIP

FP signal.

(A) FFT histograms at ZT4 and ZT9 of the

509

frequency range 0.007 to 0.05Hz,

comparing OVX females and males. Thick lines show the histogram

510

medians. P=0.00003 and P=0.0002 at ZT 4 and 9, respectively, tw

o-sample Kolmogorov-Smirnov test. (B)

511

Same as (A), for M/D vs. E. P=0.88 and P=0.02 at ZT 4 and 9, re

spectively, two-sample Kolmogorov-

512

Smirnov test. (C) Cross-validated accuracy with support vector

machine (SVM) classifier for discriminating

513

sex and hormonal state prediction ability, based on full FFT sp

ectrograms (24h), averaged over five double-

514

sided iterations for OVX, males, and females on different estro

us cycle days. Males: n=6; OVX: n=5,

515

Females: n=6; Chance level is 50%. see Figure S4 for a comparis

on with "Discriminate analysis" classifier.

516

(D) Cross validation accuracity score for M/D vs. E, using FFT

at intervals of three hours, comparing SVM

517

(black) with "Discriminate analys

is" algorithm (green), both showing a peak at ZT9-11. (E) FFT-based

518

classification, based on FFT spectrograms at ZT9-11, averaged o

ver five double-sided iterations. (F-I)

519

SCN

VIP

neuronal dynamics at ZT10-13. (F) Event rates during ZT10 to 1

3, divided to 10 minutes intervals,

520

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of cycling and OVX females. (G) Averaged event rates at ZT10-10

.5. Nonparametric Kruskal-Wallis

521

test, followed by Tukey's corrections (**p < 0.01). (H) Average

d FFT spectrograms across all states. (I)

522

Cross-validation accuracy of SVM classifiers of the discriminat

ion prediction ability across estrous cycle

523

days.

524

525

Figure 4:

CRISPR-C

as9 mediated disruption

VPAC2 and KISS1R on GnRH neurons reduces

526

estrous cycle regularity.

(A) Experimental design: viral injection to the ME with AAVret

ro, to cover all

527

GnRH neurons which project to the ME. VSs were taken for three

weeks, before and after virus injection.

528

(B) Transduction efficiency in experimental mice for KISS1R (i)

and VPAC2 (ii), three samples from each

529

animal, represented by different shades of gray. (C) Representa

tive examples of MPA histology, showing

530

KISS1R-Ab expression (orange) overlapping with GnRH neurons (Ca

s9 in green, GnRH-Ab in purple) in

531

control mice (top), but not in experimental mice (bottom), whic

h show AAVretro expression (cyan). (D)

532

Same as (C), for VPAC2. (E) The number of proestrus events identified in three weeks, before and after

533

virus injection in control (n=5)

, sgKiss1r (n=6), and sgVipr2 (

n=4) mice. (F) Estrous states appearance the

534

period of three weeks, comparing all conditions. Estrous state

distributions by percentage are available in

535

Figure S6. (*p<0.05; **<0.01, ***

<0.005; Kruskal–Wallis test, T

ukey's correction for multi comparisons).

536

537

Figure 5: Under altered light conditions, afternoon light rescu

es estrous cycle regularity and egg

538

release profile, and specific SCN

VIP

neuron activation with excitatory DREADD rescue estrous cycle

539

regularity.

(A) Light cycle manipulation experimental design. Yellow: light. Dark gray: dark. Gray

540

asterisks

indicate the time of vaginal smear collection, aiming to minimi

ze light exposure during the dark,

541

even to ambient red light. CT0

0.5L

refers to 0.5h of light at CT0. CT4

, or CT10

refers to 1h of light at CT4

542

or 10. CT0 is the activity offset in nocturnal animals (see LMA

in Figure S7) (B) Estrous stage distribution

543

under the five light conditions (VIP-Cre, n=4), P-proestrus, E-estrus, M/D- metestrus, or diestrus. (C) The

544

number of proestrus events in three weeks (mean). (D) The number of released eggs in three light conditions

545

(n= 9, 7, 4 per condition, respectively). Boxplot showing media

n (white dot), 25

(bottom), and 75

(top)

546

percentile. (E) Chemogenetic experimental design. VIP-Cre femal

es were injected with Gq expressing virus

547

(Exp, n=7) or eYFP (Ctrl, n=6), and put under four conditions,

LD, DD+CT0

0.5L

, DD+CT0

0.5L

+CT4

CNO

548

and DD+CT0

0.5L

+CT10

CNO

(CT0

L0.5

, CT4

CNO

, and CT10

CNO

correspond to 0.5h light at CT0, CNO injection

549

at CT4 and CT10, 1mg/kg, cyan). (F)

Post hoc

histology, showing virus expression, mCherry (gray, Exp)

550

and eYFP (gray, Ctrl), with c-Fos

staining (purple), ~1h after

CNO application. (G) The number of proestrus

551

events in three weeks, under the three conditions shown in E. (

I) Overall appearance of the estrous states in

552

the LD condition and in the DD+CT0

0.5L

+CT10

CNO

condition. Estrous state distr

ibutions by percentage are

553

available in Figure S8. Nonparametric Kruskal-Wallis test, foll

owed by Bonferroni corrections (*p < 0.05).

554

555

The copyright holder for this preprint

this version posted January 17, 2023.

;

https://doi.org/10.1101/2023.01.14.524075

doi:

bioRxiv preprint

The copyright holder for this preprint

this version posted January 17, 2023.

;

https://doi.org/10.1101/2023.01.14.524075

doi:

bioRxiv preprint

The copyright holder for this preprint

this version posted January 17, 2023.

;

https://doi.org/10.1101/2023.01.14.524075

doi:

bioRxiv preprint

The copyright holder for this preprint

this version posted January 17, 2023.

;

https://doi.org/10.1101/2023.01.14.524075

doi:

bioRxiv preprint