of 18
1
Dysregulated mammalian estrus cycle rescued by timed activation
of VIP
1
neurons in the circadian pacem
aker and late afternoon light exposure
2
3
Estrus cycle rescued by afternoon activation of suprachiasmatic
VIP neurons
4
5
One Sentence Summary:
Modulating and recording the activity of suprachiasmatic VIP ne
urons in freely
6
behaving mice reveals their regulation of fertility by mediatin
g the response to late afternoon light.
7
8
Anat Kahan
1
, Gerard M. Coughlin
1
, Máté Borsos
1
, Bingni W. Brunton
2
, Viviana Gradinaru
1
9
10
1
Division of Biology and Biological Engineering, California Inst
itute of Technology, Pasadena,
11
California, 91125, USA
12
2
Department of Biology, University of Washington, Seattle, Washi
ngton, 98195, USA
13
* Correspondence to V.G. (
viviana@caltech.edu
) and A.K. (
anatk@caltech.edu
)
14
15
Abstract
16
Jet lag and shift work disrupt the menstrual cycle and decrease
fertility. The circadian pacemaker, the
17
suprachiasmatic nucleus (SCN),
is known to modulate ovulation,
but the mechanism is unclear. Here we
18
explore this connection by tracking the dynamics of vasoactive
intestinal peptide (VIP)-expressing neurons
19
in the SCN in freely-behavi
ng mice. We show that SCN
VIP
activity is time-of-day-
and sex-dependent, and
20
estrous-state-dependent in late
afternoon, gating downstream ac
tivation of GnRH neurons. Afternoon light,
21
as well as specific activation of SCN
VIP
neurons, rescues estrous cycle regularity and egg release in a
nimals
22
in altered light conditions, emphasizing the role of SCN
VIP
neurons as a time-dependent light-responsive
23
switch. Our results reveal the dynamic mechanism by which SCN
VIP
neurons mediate light responses to
24
regulate estrous states and demonstrate light-induced fertility
rescue.
25
26
Keywords
: Estrous cycle; suprachiasmatic
nucleus; vasoactive intestinal
peptide; circadian rhythm;
27
GnRH; calcium imaging; fiber photometry; CRISPR
28
29
Main
30
In the mammalian estrous cycle,
fluctuations in sex hormone sec
retion occur with periods longer than a
31
day, and these infradian rhythms are coordinated with the behavioral circadian rhythm to align the fertility
32
window with social interaction and receptivity, thus maximizing
reproduction (
1
). The critical event of
33
ovulation is controlled by the h
ypothalamic-pituitary-gonadal (
HPG) axis and, in mice, is synchronized
34
with the dark phase every four or five days. In metestrus and d
iestrus, estrogen secretion from the ovary
35
increases, followed by a luteinizing hormone (LH) surge in the
late afternoon of proestrus, triggering
36
ovulation ~12 hours later. Hypotha
lamic neuronal populations su
ch as gonadotropin-releasing hormone
37
(GnRH) neurons in the medial preoptic area (MPA) (
2
), RF-amide-related peptide-3 (RFRP-3) neurons in
38
the dorsomedial hypothalamus (DMH) (
3
) and kisspeptin (KISS1) neurons in the anteroventral
39
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2
periventricular nucleus (AVPV) (
4, 5
) and the arcuate nucleus (
6
) mediate estrogen feedback and are
40
responsible for the LH surge.
41
The hypothalamic suprachiasmatic
nucleus (SCN), the circadian r
hythm pacemaker, is fundamental to
42
ovulation occurrence. Several pieces of evidence suggest that s
ignals from the SCN directly generate the
43
LH surge. First, the SCN has ne
uronal projections to relevant h
ypothalamic regions, including the MPA
44
(
7
), AVPV (
8
), and DMH (
8-10
). Second, SCN lesions cause a decrease in LH surge and estrous
cycle
45
regularity (
11-14
). Two circadian neuropeptides have been associated with the LH
surge: arginine-
46
vasopressin (AVP) and vasoactive intestinal polypeptide (VIP).
Female mice deficient in VIP show reduced
47
estrous-cycle regularity and fertility (
15
), and neurons expressing these peptides project directly or
48
indirectly to GnRH (
16-18
), Kiss1 (
6
), and RFRP-3 (
10
) neurons. Specifically, VIP receptor type 2
49
(VPAC2, encoded by
Vipr2
) is present in ~40% of GnRH neurons during proestrus, and VIP
processes
50
have been observed in a
pposition to GnRH neurons (
7, 19
). Based on bilateral differences in c-Fos
51
expression in GnRH neurons in hamsters, it was suggested that c
ircadian regulation of GnRH neurons is
52
mediated by direct synaptic input to GnRH neurons (
20
). While VIP is known to excite GnRH neurons in
53
brain slices, the estrous cycle regulation of this process is d
ebated in the literature (
16, 18, 21
), and the
54
infradian dynamics of VIP-expr
essing neurons throughout the est
rous cycle are unknown.
55
Here, we show that light information controls estrous cycle regularity through SCN
VIP
neurons. Our
in vivo
56
approach combines neuronal and physiological outcomes, which ca
n be tracked long-term. Measuring the
57
dynamics of SCN
VIP
neurons across the estrous cycle using a calcium indicator wit
h fiber photometry
58
followed by advanced machine-learning-assisted signal analysis,
we show that SCN
VIP
neuronal activity is
59
sex-specific and light-active, with increased activity in the l
ate afternoon, especially when females are non-
60
receptive. We observed a reduction in estrous cycle regularity
when VPAC2 was downregulated in GnRH
61
neurons, suggesting that this time-of-day information is gating
the estrous cycle through GnRH neurons.
62
Based on these observations, we hypothesized that afternoon lig
ht could rescue estrous cycle regularity
63
under minimal dark conditions, as indeed we found, together wit
h the rescue of egg release. Furthermore,
64
this rescue could be mimicked by specific SCN
VIP
activation in the afternoon, emphasizing the unique role
65
of SCN
VIP
neurons as a light switch. Together, this work establishes a s
olid mechanistic connection between
66
light, SCN
VIP
activity, estrous cycle, and fertility.
67
68
Results
69
Estrous cycle regularity in mice is significantly reduced by jet lag or ablation of SCN
VIP
neurons
70
In humans, disruptions in the regular light-dark cycle (e.g., d
uring jet lag) are associated with irregular
71
menstrual cycling and decreased fertility. To confirm the signi
ficance of light regularity on the mouse
72
estrous cycle, we performed a "jet lag" experiment in which we
housed female C57 mice in 12:12 hour
73
light:dark (LD) conditions, and every 4-5 days, advanced the li
ght in the room by six hours. This pattern
74
was previously found to dramatically decrease pregnancy success
in mice (
22
), and reduce general fertility
75
in humans (
23, 24
). Using cytology of daily vaginal smears over three weeks (4-5
cycles in non-jet-lagged
76
animals), we found that this light pattern decreased the regula
rity of the estrous cycle in the mice, reducing
77
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
78
mainly to increased time spent in
estrus, from 7.0±0.6 to 9.1±0
.9 days (Figure 1A-D).
79
The SCN is one of the initial brain relays for retinal response
s to light, and SCN
VIP
neurons were previously
80
identified as essential to the circadian response to light (
25-29
), forming monosynaptic connections to
81
intrinsically photosensitive retinal ganglion cells (ipRGCs) (
27, 30
) and mediating the rapid response to
82
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3
light during the dark phase (
26
). We, therefore, tested the effect of specific ablation of SCN
VIP
neurons on
83
estrous cycle regularity in female mice kept under 12:12 condi
tions. We injected adeno-associated virus
84
(AAV) to deliver Cre-dependent caspase-3 to the SCN of VIP-Cre
mice,
inducing targeted
apoptosis of
85
SCN
VIP
neurons (Figure 1E). We found that ablation significantly reduced the number of proestrus
86
repetitions, from 4.3±0.4 to 2.8±0.3 in three weeks, significantly lower than the control group (Figure 1F-
87
H). These results support a specific role for SCN
VIP
neurons in connecting light stimulus with estrous cycle
88
regulation.
89
SCN
VIP
activity is sex-specific and time-of-day-dependent
90
To characterize the temporal dynamics of SCN
VIP
neuron activity,
we used fiber photometry (FP) to record
91
bulk neuronal activity in males and females using a genetically
encoded calcium indicator, GCaMP6s, using
92
VIP-Cre x GCaMP6s mice, implanted with two 400
m optical fibers. To verify the fiber location, we used
93
light-guided-sectioning (LiGS) (
25
), a 3D histological method that we developed to determine the
precise
94
position of optical implants within deep and small brain nuclei
, such as the SCN (Figure 2A). First, we
95
tested the responsiveness of the calcium signal to the light phase transitions, zeitgeber time (ZT) 11 to 13
96
and ZT23.5 to 0.5. Using dF/F and event-rate analyses, we found
that SCN
VIP
activity is synchronized with
97
the light change (Figure S1). Having thus confirmed light sensi
tivity at the light phase transitions, we
98
recorded bulk GCaMP6s activity for 10 minutes each hour over 24
hours (Figure 2B). We determined the
99
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
2
=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 (
31
), 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 (
32
). 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
(
25
). Together, our FP results show a late afternoon decrease in S
CN
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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;
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5
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 (
38
), 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 (
40
) (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
or
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
L
) and the last at CT4 (DD+CT0
0.5L
+CT4
L
,
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 (
22
).
203
Compared to age-matched controls in 12:12 LD conditions, female
s in DD+CT0
0.5L
+CT10
L
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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6
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 (
41
).
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 (
51
).
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 (
52
).
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 (
53
) 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 (
15
)
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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;
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doi:
bioRxiv preprint
7
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 (
56
). 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 (
57
). We examined the
265
role of VIP because it was shown that the calcium activity of S
CN
VIP
neurons correlates with VIP release
266
(
26
), but we cannot rule out an additional role for GABA. Second,
recent studies have functionally (
31
) 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 (
10
).
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
(
59
). 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
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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;
https://doi.org/10.1101/2023.01.14.524075
doi:
bioRxiv preprint
8
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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
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted January 17, 2023.
;
https://doi.org/10.1101/2023.01.14.524075
doi:
bioRxiv preprint
12
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 (
25
). 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
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted January 17, 2023.
;
https://doi.org/10.1101/2023.01.14.524075
doi:
bioRxiv preprint
13
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
of
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
L
, or CT10
L
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
th
(bottom), and 75
th
(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
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted January 17, 2023.
;
https://doi.org/10.1101/2023.01.14.524075
doi:
bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted January 17, 2023.
;
https://doi.org/10.1101/2023.01.14.524075
doi:
bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted January 17, 2023.
;
https://doi.org/10.1101/2023.01.14.524075
doi:
bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted January 17, 2023.
;
https://doi.org/10.1101/2023.01.14.524075
doi:
bioRxiv preprint