of 17
1
Supplementary materials
Materials and Methods:
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Antibodies
GFP (Chicken); Cleared tissue: 1:200
Aves
GFP-1020,
RRID
AB_1608076
GFP (Goat); brain s
lices: 1:1000
abcam
ab6673
RRID
AB_305643
VIP (Rb); brain slices: 1:500
Immunostar
20077
RRID
AB_572270
c-Fos (Rb); Cleared tissue: 1:
200; brain slices: 1:500
abcam
ab209794
RRID AB_2905616
VPAC2 (Rb) ; brain slices: 1:200
Antibodies.com
A96020 (G790)
RRID AB_10695996
KISS1R (Rb) ; brain slices: 1:40 **
Proteintech
15505-1-AP
RRID
AB_10640578
GnRH (GP) ; brain slices:
1:10000
Greg Anderson, Otago
University
GA-04
Alexa Fluor 488 AffiniPure F(ab'
)2 Fragment Donkey Anti-Chicken
IgY (IgG) (H+L)*
Jackson Immune
703-546-155
RRID
AB_2340376
Alexa Fluor® 647 AffiniPure Fab
Fragment Donkey Anti-Rabbit
IgG (H+L)*
Jackson Immune
711-607-003
RRID
AB_2340626
Cy™3 AffiniPure Donkey Anti-Guinea Pig IgG (H+L)*
Jackson Immune
706-165-148
RRID
AB_2340460
Donkey Anti-Goat IgG H&L (Alexa Fluor® 488)*
abcam
ab150129
RRID
AB_2687506
Bacterial and virus strains
AAV5.hSyn.DIO.hM3D(Gq).mcherry
Addgene
44361-AAV5
AAV5.Flex.taCasp3.TEVp
UNC Vector Core
#45580
AAV5.Ef1a.DIO.eYFP.WPRE.hGH
Addgene
27056-AAV5
AAVrt.CAG.FLEX.tdTomato.WPRE
Addgene
51503-AAVrg
AAVrt.CAG.sgVipr2(A)
Glab
AAVrt.CAG.sgVipr2(B)
Glab
AAVrt.CAG.sgKiss1r
Glab
Chemicals, peptides, and
recombinant proteins
Normal Donkey Serum
Jackson Immune
017-000-121
Dimethyl sulfoxide
Fisher
D128-4
Heparin
Sigma-aldrich
H3393-100KU
Triton X 100
Sigma-aldrich
93443-100ML
TWEEN 20
Sigma-aldrich
3005
Glycine
Sigma-aldrich
G7126-100G
D-Fructose
Sigma-aldrich
F0127-1KG
-Thioglycerol
Sigma-aldrich
M1753-100ML
Clozapine N-oxide (CNO) dihydrochloride
Tocris
6329/10
Recombinant DNA an
d virus perpetra
tion components
pSpCas9(BB)-2A-GFP, Feng Zhang
Addgene
48138
2
AAV2-retro, Alla Karpova and
David Schaffer
Addgene
81070
Neuro-2a cells
ATCC
CCL-131
HEK293T cells
ATCC
CRL 3216
QuickExtract DNA Extraction Solution
Lucigen
QE09050
NEBuilder HiFi
New England Biolabs
E2621
polyethylenimine
Polysciences
24765-1
Iodixanol, Optiprep
Sigma-Millipore
D1556
Software and algorithms
ICE analysis
Synthego
ImageJ, version 2.0.0-rc-69/1.52n
NIH, open source
https://fiji.sc/
Adobe Illustrator, 24.0.2 (64-bit)
Adobe
https://www.adobe.com/pr
oducts/illustrator.html
Matlab R2020b, R2018a
MathWorks Inc.
https://www.mathworks.co
m/products/matlab.html
Imaris 9.2.0
Oxford Instruments
https://imaris.oxinst.com/
Zen (LSM 880, 2.3 lite)
Zeiss Microscopy
https://www.zeiss.com
BioRender
BioRender
https://biorender.com/
Custom code to analyze and ge
nerate figures
This paper
GradinaruLab/SCN-VIP-
estrous-cycle: Anat Kahan,
Jan 2023 (github.com)
Other
Optical fiber, 400 μm diameter, 7 mm long
Doric Lenses
MFC_400/430-
0.48_7mm_ZF1.25_FLT
Mono Fiberoptic Patch c
able (fiber photometry)
Doric Lenses
MFC_400/430_0.48_2m_F
C_ZF1.25_FL
Double patch cable (optoge
netics)
Doric Lenses
BFP(2)_400/430/1100_0.4
8_1.5m_FCM*-
2xZF1.25(F)
Table S1: Detailed reag
ent and resources.
* All secondaries were used at 1
:200 for cleared tissue, and 1:
1000 for 70/100
m brain slices
**This ab was incubated for two over-nights in 4°c.
sgRNA crRNA sequence
Forward primer
Reverse primer
Vipr2-B GGGCATCCGAATGACCCACC TTGACTCACCCAGGAAAGCC TTGGAAATGGGCA
GCAGAC
T
Kiss1r-B AGCGTTGGACGGAGCCCACC CA
GAGGAGCCTCTTTCCAGC TTCCTGACTTGG
ACCCCAGA
Table S2: related to figure 4. crRNA sequences and associated p
rimers for Genetic knockout in GnRH neurons using
CRISPR
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).
The usage of Ai162 line, when crossed to a Cre line, allows sup
pression of GCaMP6s expression using a
doxycycline diet. Therefore
,
for fiber photometry recording, the diet was given to the breed
ing pair and the
3
offspring until 2-4 weeks before surgeries, to prevent toxicity
or developmental issues. For optogenetic
experiments, VIP-IRES-Cre line was crossed to Ai132 (ChR2, JAX,
24109). For GnRH neurons gene
editing, GnRH-Cre line (Gnrh1-Cre, JAX, 21207) was crossed to R
osa26-Cas9 (JAX, 24858). Animals
were group housed (2-4 per group)
whenever possibl
e to ensure a
regular estrous cycle. Mice were single-
housed for FP experiments that required 24/7 recordings and opt
ogenetic experiments. Females that were
single housed for calcium recording received a handful of fresh
male mice bedding every cage change to
ensure regular estrous cycles. Mice had a running wheel or a cl
eared tube for enrichment in their home
cage. For optogenetics, mice were connected with two-branched p
atch cables, so runnin
g wheels/tubes were
impossible. Mice were kept at 12:12 light cycle, unless stated
otherwise. The ambient room light intensity
was 4.49E14 photons/cm
2
/sec (150 lux). Light at ‘lux’ units was measured with the Ligh
t Meter smartphone
app (‘My mobile Tools Dev’). Light power (mW) was measured with
Thorlabs S120C photodiode. The
energy units were divided by the sensor dimensions, 0.94 cm
2
. Unit conversion from mW/cm
2
to
photons/cm
2
/sec were calculated using:
௣௛௢௧௢௡௦
∙퐸ൌ푛
௣௛௢௧௢௡௦
௛௖
, where
is Plank constant, c is the
speed of light, and
is the light wavelength (at maximum). Mice were used until 10
months old to ensure
a regular estrous cycle (
1
), and were age-matched for each experiment. Animals had
ad libitum
access to
food and water.
FP recording
,
light manipulation, optogenetic and chemogenetic experiments w
ere performed in rat cages
(40 x 34 cm). For FP, the lid was modified to have a hole in th
e middle for the patch cable.
Light manipulation experiments with PIR detection and/or optoge
netic manipulation were performed in rat
cages, with a 10-inch diameter Plexiglas cylinder, 34 cm in hei
ght, to ensure sufficient PIR detection. Mice
were group housed for light manipulation experiments. For optog
enetic experiments, mice were single-
housed due to the patch cable. Other light manipulation experim
ents (cohort 1 of DREADD, C57) were
performed in regular mice cages.
Animal husbandry and experiment
al procedures involving animal s
ubjects were conducted in compliance
with the Guide for the Care and Use of Laboratory Animals of th
e National Institutes of Health and
approved by the Institutional Animal Care and Use Committee (IA
CUC) and by the Office of Laboratory
Animal Resources at California In
stitute of Technology under IA
CUC protocol 1739. Mice were excluded
from the entire FP experiment i
f there was no dynamic photometr
y 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 (
2, 3
), and improvement was made based on 2D or LiGS histology. A cr
aniotomy hole was drilled
above the target. Virus injecti
on and implantations were perfor
med 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 an
d -5.6 mm from the brain, left and
right sides, at 13 degrees, 2 x 300 nl each side.
4
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 (
4
), prepared as described i
n the following section.
AP -1.4 mm, ML ± 0.3 mm, DV -5.65 and -5.4 mm from the brain, l
eft 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
l microsyringe (NanoFil, World Precision Instruments, WPI) by a
n 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, Can
ada) were firmly mounted to a
stereotaxic holder. Two fibers were implanted to improve the pr
obability of successfully targeting the
structure. A thin layer of Metabond (
Parkel
) was applied to the skull surface to secure the fiber. In addi
tion,
a thick layer of black dental cement (JET denture repair powder
and liquid) was applied to secure the fiber
implant and to prevent interfe
rences between the excitation lig
ht to light-sensitive targets that are not the
SCN.
Extended details about viruses and implants can be found in Tab
le 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 an
d sustained-release buprenorphine
at 1mg/kg SC. The mouse was then
anesthetized with 1-5% isoflur
ane in an induction box followed by
maintenance in a nose cone and w
as maintained on a heating pad
throughout the surgery. A small dorsal
midline incision was made over the abdomen. The abdominal cavit
y was entered via a blunt puncture
through the abdominal wall. The ovary was dissected. The fat pa
d and tissue were returned to the abdominal
cavity, and the abdominal wall was closed with 4-0 absorbable m
ultifilament 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 monofila
ment 4-0 suture material in an interrupted
pattern. Before closure, bupivacai
ne (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 sme
ars for at least 10 days, showing either
diestrus or metestrus states.
All m
ice 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 f
or at least five days postoperatively for
pain. Mice were allowed a minimum of 14 days for surgical recov
ery 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, Ne
uro-2a cells (ATCC CCL-131) were
transfected in duplicate with PX458 containing sgRNAs of intere
st, 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 analyz
ed for editing efficiency using ICE
analysis (Synthego). Only sgRNA sequences that yielded >70% edi
ting 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.
5
For
in vivo
sgRNA delivery, pAAV.U6-sgRNA(Sa
pI)-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 crR
NA insertion into MluI-digested
pAAV-CAG-mRuby2 (
5
), using NEBuilder
HiFi (New England Biolabs, E2621). crRNA sequences were inserte
d by annealing complementary oligos
containing overhangs, followed by ligation into SapI-digested p
AAV.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 (
6
) capsids through triple trans
ient transfection of HEK293T
cells (ATCC, CRL 3216) with polye
thylenimine (Polysciences, 247
65-1), followed by cell lysis and
purification over iodixanol (Optiprep; Sigma-Millipore, D1556),
as previously described (
7
). The AAV2-
retro rep-cap plasmid was a gift from Alla Karpova and David Sc
haffer (Addgene ID: 81070). Viral titers
were obtained through qPCR, using a linearized genome plasmid a
s 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 fluor
escence 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 elsewhe
re (
8
). Briefly, o
ur system employed a
490 nm LED for fluorophore exc
itation (M490F1, Thorlabs; filter
ed with FF02-472/30-25, Semrock) and
a 405 nm LED for isosbestic excitation (M405F1, Thorlabs; filte
red with FF01-400/40-25, Semrock),
which were modulated at 211 Hz and 531 Hz, respectively. Two sy
stems 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 emissi
on signal from isosbestic
excitation, which was previously shown to be calcium-independen
t for GCaMP sensors (
9, 10
), was used
as a reference signal to account for motion artifacts and photo
bleaching. Emitted light was collected via
the patch cable, collimated, filtered after passing through a f
ocusing 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 photor
eceiver (Model 2151, Newport). Ph
otoreceiver signals were demodulated
into GCaMP and control (isosbestic) signals, digitized (samplin
g rates: System 1: 382 Hz; System 2: 6
Hz), and low-pass filtered at 25 Hz using a second-order Butter
worth filter with zero phase distortion. A
least-squares linear fit was applie
d 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 divid
ed by the fitted 405 nm signal to
calculate dF/F values.
Behavioral assays
Estrous Cycle Stage Identification:
Estrous cycle stage was recorded
following established protocol
s (
11
).
Briefly, vaginal smears (VS) were collected with a 100 or 20
l pipette holding 15
l saline at ZT10-14. In
cases where the room was entered for injections or light stimul
ation, VSs were taken at the time of
interference to reduce entrainments. VS were flattened and allo
wed to dry on a microscope slide (Adhesion
Superfrost Plus Glass Slides, Brain Research Laboratories) and
stained with cresyl violet dye (0.1%, Sigma-
Aldrich, C5042-10G). Smears were then analyzed using light micr
oscopy. 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 in
creased amount of leukocytes, wi
th the presence of cornified epithelial cells
6
and some nucleated epithelial cells. Diestrus is characterized
by mostly leukocytes, with low presence of
cornified epithelial cells and nuc
leated epithelial cells. We d
efined the number of proestrus events based
on the number of identified proestrous events, even if a full c
ycle was not detected during the monitored
period.
OVX and hormonal replacement:
OVX was verified by having a VS profile of diestrus or metestru
s. At
least four weeks after OVX, sex hormone stimulation was perform
ed as follows; e
stradiol was administered
at 10 μg and progesterone at 500 μg in 0.05 ml of sesame oil de
livery s.c. at ZT8, which has been shown to
induce sexual receptivity. These estradiol levels are comparabl
e to physiological proestrus peak levels (
12
).
Locomotor activity (LMA) detection:
Mice were placed in a rat cage (40 X 34 cm). A PIR motion detec
tor
(COMPASS (
13
)) was placed 35 cm above the cage bottom, and a Plexiglas cyli
nder was placed in the
middle (10-inch diameter) to ensure even detection of mouse act
ivity. Mice were placed with a tube used
for handling to reduce anxiety (
14
).
Chemo- and Optogenetics:
After recovery from the surgery, mice were moved to the behavio
ral room with
the light cycle used for the experiment and handled for at leas
t 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. Follo
wing the first baseline session, the light
cycle was changed to DD, with additional light stimulation at C
T0 (DD +CT0
0.5L
), to preserve their
locomotor rhythm as much as possible. At either CT4 or CT10, st
imulation 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) (
15
). In addition to the black dental cement, we used two layers
of black heat-shrink tubes to prevent interferences 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 pe
r 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 ZT14) and examined c-Fos and ChR2
or hM3Dq expression using IHC.
During all light and neuronal manipulation studies, vaginal sme
ars (VSs) were taken daily at ~ZT10, except
for opto- and chemogenetic experi
ments, in which VSs 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 week
s. 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 expe
riment due to severe weight lo
ss (>10%). For light and SCN
VIP
activation
experiments, females that were not affected by the reduced ligh
t conditions were not considered as
‘rescuable’, and therefore were excluded (6/41).
Released egg collection
: Ovulating eggs were collected from the oviduct the day after
ovulation, within 24-
30h after the detected proestrus state. Cumulus-oocyte complexe
s were isolated in M2 media (Sigma,
M7167) supplemented with 0.3% hyaluronidase (Sigma, H3506) to d
etermine the number of eggs ovulated.
7
LiGS 3D histology
LiGS tissue preparation:
LiGS histology of implanted mi
ce was performed as described pre
viously (
16
):
after perfusion (20 ml 1× PBS followed by 20 ml 4% PFA), the im
plant was kept intact; the skin and the
lower jaw were gently removed. The remaining skull, including b
oth 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, Sak
ura Finetek) and were frozen with
an ethanol/dry-ice bath (−78°C)
. The brain was
positioned such
that the optical impla
nt 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 c
ube 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 pr
ocess created a large, stable OCT cube that
included the sample and the coupled LED. The sample was then cu
t 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 bot
tom in 50–
100um steps with the LED turned on.
To ensure a reproducible li
ght 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, se
ctioning was continued in small steps (20–50
m) until a shaded area was seen i
n 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 adapte
d from the iDisco protocol with
out 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 a secondary antibody in P
TwH/3% donkey serum at 37°C for 5–7
days. Lastly, samples were washed
with PTwH at RT until the nex
t day.
LiGS Clearing
: Samples were cleared using the SeeDB protocol at RT (
17
).
Thin slice staining:
After perfusion (20 ml 1× PBS followed by 20 ml 4% PFA), brain
sections (70 or 100
m, 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 inc
ubation with primary antibodies in the
blocking buffer solution at 4°C overnight. The next day, sectio
ns 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. Ne
xt, sections were washed as described above
and mounted on glass microscope slides (Adhesion Superfrost Plu
s Glass Slides, Brain Research
Laboratories). After the sections were completely dry, they wer
e cover-slipped after applying mounting
media (VECTASHIELD® PLUS).
Extended information about IHC
materials can be found in Table
S1.
8
Histological imaging
Histological images were obtained with Zeiss 880 confocal micro
scope 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 an
d 9.9.1), with a diameter of 15
m, for
each channel separately. In addition, the “Statistics” toolbox
of Imaris was used to identify overlapped
populations (<10
m). For each animal, three brain slices which included the MPA
were quantified, and
the injection quality was validat
ed by observing the injection
site (Figure S6).
Data analysis
Light detection
LMA:
Following PIR motion detection, the mean activity and onset wer
e calculated using an angular
presentation of each 24-hour period, using the “CircStat” toolb
ox for circular statistics (Matlab (
18
)). 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
used [30]. When z-
scored data was presented, we used:
푑퐹 퐹
ൌ퐹െ푚푒푑푖푎푛
푚푎푑
⁄ሺ
(where ‘mad’ is the median
absolute deviation, and
is the background signal during the dark phase). The event rat
e 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 the 10 minutes/h sess
ions, and 0.4 for the ZT10-13 recording,
justified by the reduced ability of ‘event rate’ analysis to id
entify low amplitude frequencies, shown by the
FFT classification analysis. For 10 minutes/h sessions, data wa
s collected starting at ZT8 for 24h. For the
analysis, data were 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 pha
se and the following dark phase. In the case of
proestrus, ovulation occurs during the dark phase, and the foll
owing light phase should be considered as
estrus. For male-to-female comparison, female data was taken fo
r 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 one set of freq
uency intervals: [0.0033 0.007; 0.007 0.05;
0.05 0.1; 0.1 0.25; 0.25 0.45; 0.45 1.0; 1.0 1.35] Hz. The lowe
r limit was set to 0.0005 for the ZT10-13
recording, due to the longer time frame. FFT autocorrelations w
ere calculated for each FFT power interval
over the coarse frequency intervals (set (a),‘autocorr’, Matlab
). See Figure S3 for an example of the FFT
approach on synthesized data.
For classification, a spectrogram of the fine frequency interva
ls was prepared for each session. Each session
was tagged by sex and by hormonal state. Prior to machine learn
ing, the spectrogram dimensions were
reduced by (1) choosing only frequency ranges between 0.003 to
1 Hz (the first six inte
rvals), (2) applying
PCA (pca, ‘Matlab’), i.e. 24 h x 6 frequencies intervals were r
educed 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 “s
upport vector machine” (SVM, ‘fitc
ecoc’, Matlab), or a “discriminant analysis”
(‘fitcdiscr’, Matlab) classifier, followed by prediction (‘pred
ict’ Matlab). In each classification, the
prediction rate was calculated for the ability to predict each
of the two states, 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.
Extended information about Soft
ware and algorithms can be found
in Table S1.
9
Figure S1, related to Figure 2
:
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 transiti
on from dark to light (ZT23.5 to 0.5).
(A) SCN
VIP
FP activity at ZT11-13 (i) Representative examples of FP record
ings 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 male
s and females at ZT 11-12
and ZT 12-13 (mean±sem, with individual values shown). Event ra
tes 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 i
n 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. a
t ZT12-13, males and females, r
espectively. (B) SCN
VIP
FP activity at ZT23.5-0.5. (i)
Averaged responses (mean±sem, sem in gray, females: n = 7, male
s: n = 5, averaged for at least three repeats each).
(ii) Mean dF/F, showing no diffe
rences between males and female
s and a significant increase from dark to light. (iii)
Event rates show no differences between males and females and s
ignificantly increase from dark to light.
Nonparametric Kruskal-Wallis test, * p <0.05.
10
Figure S2, related to Figure 2: Circadian changes in SCN
VIP
signal are sex-dependent but not estrous-cycle
dependent.
(A) Representative examples of 10
-minute-per-hour recording, ov
er 24 hours, from a female (left) and a
male (right) mouse. (B) DF/F (top) and event rates (bottom) ove
r 24h of recording (p values compare females, n=6
(red) vs. males, n=6 (blue), at least 3 repeats each. (C) Compa
rison of median dF/F (top) and median event rate
(bottom) between males and females over the dark and light phas
es. (D) DF/F over 24h of recording across estrous
states (n=8, each state is represented at least three times in
each female). (E) DF/F averaged over dark and light phases.
For clarity, significance is ma
rked only between adjacent perio
ds. (F) Parabolic fits to median dF/F along ZT2-11.
(G) Averaged event rates over two hours at different points dur
ing the day, showing no significant differences between
estrous states. Data was assigned
relative to the proestrus day
, therefor M/D states were identified either one or two
days before proestrus (M/D P-2 and M/D P-1), as well as two day
s after proestrus, which were either E or M.
Nonparametric Kruskal-Wallis test, followed by Tukey's correcti
on when multi-comparison was done (*p<0.05;
**<0.01; # < 0.005).
11
Figure S3, related to Figure 2: FFT spectrogram creation approa
ch using artificial data.
(A) Artificial FP data
is a combination of two sine signals, for ZT0-11 and ZT12-23 se
parately, at frequencies of 0.016 and 1.5Hz, with
amplitudes of 10 and 1, as well as 0.02 and 1.0 Hz, with amplit
udes 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 c
ontributing frequencies, 0.016,
0.02, 1.0, and 1.5 Hz. (D) Integrated FFT over different
frequency intervals.
12
Figure S4, related to Figure 3: Cross-validated accuracy with “
Discriminate analysis” algorithm,
using the full
24h FFT spectrograms (left) or
ZT9-11 (right). The accuracy val
ues show similar prediction abilities to the SVM
algorithm.
Figure S5, related to Figure 3. Activity analysis of ZT10-13 FP
data, including OVX induction with sex
hormones
. (A) Averaged dF/F at ZT10-13 a
cross estrous states. (B) Quant
ified dF/F (top) and event rates (bottom),
including OVX induction with sex hormones. *p<0.05; Kruskal–Wal
lis test, Tukey's correction for multi-
comparisons.
13
Figure S6, related to Figure 4.
Validation of GnRH-Cre x cas9 inj
ection site and VPAC2 antibody
and estrous
state distributions
. (A) The injection site, the median eminence (ME). (B) VPAC2 A
b validation based on expression
in the SCN. (C) Estrous states distributions over three weeks,
before and after injection, control (Ctrl, n=5), sgVipr2
(n=4), and sgKiss1r (Exp, n=6).
14
Figure S7, related to Figure 5
.
LMA under different lig
ht conditions of VIP-cre female mice
. (A) LMA under
each light condition of one of th
e cages. Gray asterisks indica
te VS collection time window. For DD condition, ambient
red light was turned on for 10 minutes. (B) Daily onset (left)
averaged (middle) LAM (n cages= 2), presented over
days. Each cage had three females and a running wheel for enric
hment.
15
Figure S8, relate to Figure 5. The
estrous-state distributions
of
SCN
VIP
chemogenetic acti
vation experiment.
(A) The experimental design (sam
e as Figure 5).
(B) Estrous sta
tes distributions over three weeks, control (Ctrl, n=6)
and experimental (Exp, n=7).
16
Figure S9, related to Figure 5. SCN
VIP
optogenetic activation in the l
ate afternoon is insufficient t
o 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+CT0
L
+CT4
opto
(447nm excitation at CT4) and DD+CT0
L
+CT10
opto
(447 nm excitation at CT10,
blue, CT0
L
is light for 0.5h at CT0). (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 impla
nt. 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 n
euronal activation, showing c-F
os expression ~1h after illumination with 447 nm
laser, at 3D (top) and 2D (bottom). (iii) VIP-Cre x GFP mouse w
ith 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
ZT14 for 1h.
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