SSRN Id4555734

Highlights
 BOLD responses were sensitive to photoperiod over the whole ewe brain
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 Negative BOLD responses were found at the beginning of short days in the hypothalamus
 Hypothalamic rCBV, tNAA and Glx concentrations changed significantly depending on
photoperiod
 Photoperiodic functional and metabolic changes in the hypothalamus promote neuronal
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proliferation and differentiation associated with adult neurogenesis
 Sheep is a promising model for the assessment of natural neurogenesis
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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4555734
Multiparametric MR Evaluation of the photoperiodic regulation of hypothalamic structures
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Nathalie Just1,2 *, Pierre Marie Chevillard1, Martine Batailler1, Jean-Philippe Dubois1, Pascal Vaudin1,
Delphine Pillon1, Martine Migaud1
Abbreviated title: BOLD fMRI and MRS of the sheep
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Conflicts of Interest : None
Acknowledgements : This study was funded by a grant from Agence Nationale de la Recherche (ANR-
16-CE37-0006-01) to Martine Migaud. Nathalie Just received research funding from the Lundbeck
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Foundation (Experiment grant, grant nr. R370-2021-402)
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1. INRAE Centre Val de Loire, UMR Physiologie de la Reproduction et des Comportements CNRS,
IFCE, INRAE, Université de Tours, 37380 Nouzilly, France
37380 Nouzilly France
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2. Danish Research Centre for Magnetic Resonance (DRCMR), Hvidovre, Denmark
ORCID: Nathalie Just: https://orcid.org/0000-0002-9401-0669; Martine Migaud: 0000-0002-9496-

0517
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*Corresponding author
Dr Nathalie Just
Danish Research Centre for Magnetic Resonance (DRCMR), section 714

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Center for Functional and Diagnostic Imaging and ResearchCopenhagen University Hospital -
Amager and Hvidovre Kettegård Allé 30
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2650 Hvidovre
Denmark
Office phone: +45 38626205 Email:nathaliej@drcmr.dk

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Keywords: Ewes, hypothalamus, 1H-MRS, Adult neurogenesis, photoperiod

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ABBREVIATIONS:
Alanine (Ala), Aspartate (Asp);Cr (Creatine); γ-amino butyric acid (GABA), Glucose (Glc),
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Phosphocholine (PCho), Glutamate (Glu), Glutamine (Gln), N-Acetyl-Aspartate (NAA), N-
Acetylaspartylglutamic acid (NAAG), Total NAA (tNAA = NAA + NAAG),; myo-Inositol (mI), Glutamine
+ Glutamate (Glx), Total Choline (tCho= PCho + GPC).
AN: Adult Neurogenesis
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ANOVA: Analysis of variance
ARH: Arcuate Nucleus of the hypothalamus
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BOLD –fMRI: Blood Oxygen Level dependent functional MRI
CRLB: Cramér-Rao lower bounds
CSF: Cerebro-Spinal Fluid
DSC-MRI: Dynamic Susceptibility Contrast MRI
FASTESTMAP: Fast, Automatic Shim Technique using Echo-planar Signal readouT for Mapping Along
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Projections
GM: Grey Matter
GnRH: Gonadotropin-Releasing hormone
HIF: Hypoxia -inducible factor 1α
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HYP: Hypothalamus
LASER: localization by adiabatic selective refocusing;
LCModel: Linear Combination Model
LD or LP: Long days or Long photoperiod
SD or SP: Short days or Short photoperiod
ME: Median Eminence
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MRI: Magnetic Resonance Imaging

MRS: Magnetic Resonance Spectroscopy
MPRAGE: 3D magnetization prepared rapid gradient echo
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NEX: Number of excitations

SGZ: Subgranular zone
STEAM: Stimulated Echo Acquisition Mode
SVZ: Subventricular zone
SCN: Suprachiasmatic nuclei
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VASO: Vascular space Occupancy

VOI: Voxel of Interest
WM: White Matter
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ABSTRACT
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Most organisms on earth, humans included, have developed strategies to cope with environmental
day-night and seasonal cycles to survive. For most of them, their physiological and behavioral
functions, including the reproductive function, are synchronized with the annual changes of day
length, to ensure winter survival and subsequent reproductive success in the following spring.
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Sheep are sensitive to photoperiod, which also regulates natural adult neurogenesis in their
hypothalamus. We postulate that the ovine model represents a good alternative to study the
functional and metabolic changes occurring in response to photoperiodic changes in hypothalamic
structures of the brain.
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Here, the impact of the photoperiod on the neurovascular coupling and the metabolism of the
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hypothalamic structures was investigated at 3T using BOLD fMRI, perfusion-MRI and proton magnetic
resonance spectroscopy (1H-MRS). A longitudinal study involving 8 ewes was conducted during long
days (LD) and short days (SD) revealing significant BOLD, rCBV and metabolic changes in
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hypothalamic structures of the ewe brain between LD and SD. More specifically, the transition
between LD and SD revealed negative BOLD responses to hypercapnia at the beginning of SD period
followed by significant increases in BOLD, rCBV, Glx and tNAA concentrations towards the end of the
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SD period. These observations suggest longitudinal mechanisms promoting the proliferation and
differentiation of neural stem cells within the hypothalamic niche of breeding ewes.
We conclude that multiparametric MRI studies including 1H-MRS could be a promising non-invasive
translational technique to investigate the existence of natural adult neurogenesis in-vivo in
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gyrencephalic brains.
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INTRODUCTION:
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Adult neurogenesis (AN) is the process of continuously generating new cells (neurons and glial cells)
that can functionally integrate pre-existing circuits in the mammalian brain throughout life. AN
recapitulates the developmental process of embryonic neurogenesis from proliferation to
differentiation, migration, axonal and dendritic development and finally synaptic integration of new
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born neurons. This process is known to occur in specific areas of the brain, namely the sub-granular
zone (SGZ) of the dentate gyrus, the subventricular area lining the lateral ventricles (SVZ) and the
tuberal hypothalamus (Doetsch et al. 1997; Kempermann G, 2002; Lledo et al., 2006; Yoo et al.,
2018;, Migaud et al., 2015; Butruille et al., 2018).
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The hypothalamic neurogenic niche has driven less attention compared to the other neurogenic
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niches but its implication in metabolic and eating disorders as well as reproductive functions
represents a major area of research (Pellegrino et al., 2018; Batailler et al., 2014; Knobloch et al.,
2016). While structural and functional aspects of AN have already largely been investigated, little is
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known about functional and metabolic regulation of hypothalamic AN although recent studies
suggest that the metabolism ensures life-long addition of new neurons in the mammalian brain
(Migaud et al., 2010). Hypothalamic structures of the brain make the link between the endocrine and
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the central nervous system and are responsible for the maintenance of the body’s internal balance.
They play a crucial role in several functions such as the regulation of appetite and body temperature,
the control of reproduction and sexual behavior, the release of hormones, the control of daily
physiological cycles and of emotional responses (Hofman et al., 1992). These important structures
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are densely populated with cells that are able to regulate glucose (Glc) availability by sensing various
metabolites (glutamate (Glu) and γ-aminobutyric acid (GABA)) to regulate energy balance, among
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which astrocytes, tanycytes and microglia (Garcia- Caceres et al., 2019).

Neuroimaging (Positron Emission Tomography (PET) and magnetic resonance imaging (MRI)) studies
both in rodents and in the human contributed to the in-vivo evaluation of the relationships between
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altered feeding behavior or reproductive activity and hypothalamic responses (Kamali et al., 2020) as
well as the evaluation of functional connectivity between the hypothalamic structures and other
brain areas (Le et al, 2020). These investigations led to a better knowledge of hyperemic
mechanisms, dopaminergic circuits, reward-based theories, Gonadotropin-Releasing Hormone
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(GnRH) neuronal system, etc… within various hypothalamic nuclei surrounding the third ventricle of
the brain, thus providing insights into the regulation of blood-brain and blood-CSF exchanges. Such
studies may provide new ways for the evaluation of risk factors for eating and reproductive disorders
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and for the design of prevention strategies for various neurodegenerative and metabolic diseases.
However, functional studies remain difficult to implement and can lead to non-specific results due to
the variety of hypothalamic nuclei that have different functions and also due to limited spatial
resolution. Appropriately defining a specific, repeatable and reproducible functional paradigm
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remains challenging. In addition, these hypothalamic nuclei are generally small, located deep in the
brain and close to the 3V, which can induce CSF-artefacts (Thomas et al., 2013). Despite the small
size and depth of hypothalamic structures, non-invasive approaches such as proton MR spectroscopy
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(1H MRS) have been also conducted successfully in rodents and humans to explore normal and
altered hypothalamic metabolism (Lizarbe et al., 2019, Just et al., 2011a, Just et al., 2011b, Joers et
al. 2017). Notwithstanding these pioneering investigations, the hypothalamus remains an
underexplored structure of the brain and novel strategies are needed for a more accurate
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understanding of the mechanisms underlying alterations of the functions it controls (Just et al.,
2022).
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Sheep belong to a large and long-lived mammalian species with long development and life
expectancy and possess a gyrencephalic brain with similar structures to the human brain such as the
hypothalamus (Fig.1 and Fig S1). Studies of the hippocampal, olfactory and hypothalamic AN in sheep
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revealed distinctive features in the dynamics of neuronal maturation compared to rodents and
appears to exhibit longer maturation time for new neurons (Brus et al., 2013; Migaud et al., 2011).
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AN of the sheep hypothalamus is strongly modulated by photoperiod (Butruille et al., 2018, Batailler
et al., 2016). This physiological response of organisms to the ratio of the day to the night length
represents a developmental strategy of conservation, having a direct impact on reproduction by
alternating periods of sexual rest and sexual activity. High levels of hypothalamic cellular
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proliferation (Butruille et al., 2018, Brus et al., 2013; Batailler et al,, 2016) are associated to short
photoperiod (short days (SD), from August till January in Ile de France ewes) whereas basal levels of
hypothalamic cellular proliferation are observed during long photoperiod (long days (LD), from April
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till July) therefore representing a natural AN phenomenon. We believe that the investigation of a
potential functional and metabolic link between photoperiod and AN using non-invasive biomarkers
such as BOLD responses and metabolite concentrations could bring new insights to the in-vivo
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assessment of the existence of AN. Moreover, the sheep model used in the present study may
represent a promising alternative animal model for translational purposes.
In the present work, BOLD-fMRI, perfusion-MRI and 1H-MRS were conducted longitudinally in the
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hypothalamic brain regions of adult ewes at 3T to investigate vascular and metabolic changes due to
the photoperiod and its association with AN.
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2. MATERIALS AND METHODS:
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2.1 Animals:
This study was approved by the Val de Loire animal experimentation ethics committee (CEEAVdL) by
the guidelines of the French Ministry of Agriculture for animal experimentation and European
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regulations on animal experimentation. This study was not preregistered. All experiments conform
with the ARRIVE guidelines and were performed following the regulations regarding animals
(authorization N° 22353 of the French Ministry of Agriculture under EEC directive). All experiments
were performed in 8 adult Ile-de-France (IF) ewes aged 3 to 4 years old, gathered under standard
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husbandry at the INRAE Val-de-Loire research center (Nouzilly, Indre-et-Loire, France, 47°33′00.8“N
0°46′55.3“E). The IF breed shows seasonal reproductive cycles that are well characterized
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(Chanvallon et al., 2011, Zarazaga et al., 2003). The experimental facilities were approved by the local
authority (agreement number E37–175–2). All animals were fed daily with hay and pellets and
had ad-libitum access to water. No randomization procedure took place. No exclusion criteria were
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predetermined and no animals were excluded or died during the experiments. Animals were
examined at two time points (LD1-T01, LD2-T02) from May till July during long days (LD) and at two
time points (SD-T01, SD-T02) from late September till November during short days (SD) (Fig.2). These
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time points during LD and SD corresponded to basal and increased levels of AN respectively (Migaud
et al., 2015; Butruille et al., 2018). Eight ewes underwent 1H-MRS, Blood Oxygen Level dependent
functional MRI (BOLD- fMRI) and perfusion MRI. Time points were spaced to allow for recovery from
anaesthesia. Blood samplings were performed twice per week from March till November to follow
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each ewe’s hormonal status by measuring plasmatic progesterone concentration. Briefly, after
catheterization of the jugular vein, approximately 5 ml of blood from the jugular vein and always at
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the same time of day (9:00 h) were drawn in vacutainers. After centrifugation, blood plasma was
transferred into smaller tubes and deep-frozen. At the end of November, once all the blood samples
had been collected, plasma progesterone was assayed by the radioimmunoassay method described
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by Terqui and Thimonier (1974). When progesterone concentration was lower than 0.75 ng·mL-1 of
plasma, the ewe was considered to be in the follicular phase of the cycle or in anovulation. As
expected, from September to December, which corresponds to the breeding period, all ewes were
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cycling (show cyclic progesterone concentrations) (Fig S2.A) indicating an increased sensitivity of the
reproductive axis to short days.
The day before MRI experiments, ewes were transported to the MR facility. Care was taken to always
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provide company to individual animals to avoid stress since sheep are a gregarious species. Each
animal was fasted 24-hours prior to intubation. Following immobilization, the sheep was intubated
after intravenous administration of a mixture of ketamine and xylazine (20 mg/kg). Each ewe was
transported to the MRI room, installed prone on the MRI bed (Fig. 2) and anaesthesia was
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immediately switched to 1.5-2% isoflurane (Just et al., 2021) in medical air through a respirator
(Aestiva, GE Healthcare, Datex-Ohmeda, USA). The respirator allowed continuous control of
respiration rates. An oximeter was attached to one of the hind-paws allowing for the control of the
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partial pressure of oxygen and heart rate. The temperature was controlled through MRI-compatible
rectal probe. The duration of each MRI session was 150 minutes for each animal. No rumenostomy
was performed. Frequent checks by a veterinary were performed during experiments. Animals were
removed from the MRI when intra-abdominal pressure was estimated to be too high. All ewes were
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scanned between 8 am and 3 pm over 3 days per time point under the exact same conditions.
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2.2 Magnetic Resonance acquisitions:
MR imaging was conducted on a 3T whole body MR Scanner (Siemens Verio, Erlangen, Germany)
with a large 4 channel flex coil surrounding the entire head. Following the acquisition of pilot images,
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T2-weighted multislice images were acquired covering the entire sheep brain for further slice
positioning. Structural images were acquired using the T1-weighted 3D magnetization prepared rapid
gradient echo (MPRAGE) sequence (TR/TE/TI=2500/318/900 ms; Flip angle = 12; NEX= 2; FOV=192 x
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192 mm2, Matrix size = 384 x 384; Voxel size = 0.5 x 0.5 x 0.5 mm3).
2.2.1 Functional magnetic resonance imaging studies
Blood-Oxygen Level Dependent functional MRI (BOLD-fMRI) was conducted using a multi-slice
single shot EPI sequence (TR/TE= 2970/30 ms; flip angle = 90°; FOV = 188 x188 mm2; Matrix= 72 x 72;
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Slice thickness = 3 mm; slices = 20). 7% CO2 was delivered through tubing directly connected to the
intubation system. The paradigm of stimulation consisted of 3 cycles of 60 s OFF-60 s ON periods for
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a total acquisition time of 11 minutes.
Perfusion-MRI measurements: Dynamic Contrast susceptibility MRI (DSC) MRI measurements were
performed using a single-shot gradient echo EPI sequence (TR/TE= 1500/21 ms; flip angle = 90°; FOV:
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192 x192 mm2 Matrix size = 64 x 64; 19 slices; Slice thickness = 1 mm) using a short bolus of 0.1
mmol/kg of DOTAREM (Guerbet, Roissy, France) followed by a saline flush using a power injector
(Medrad). The contrast agent was injected after 60 s of acquisition at 5ml/s. 60 to 80 time points
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were acquired.
2.2.2 Image processing:
Functional MR images were processed with SPM12 software (Statistical Parametric Mapping,
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London, UK). After pre-processing, images were co-registered to structural MPRAGE images and
normalized to the in-house developed sheep brain template (Ella et al., 2017). Finally, images were
spatially smoothed using a 6 x 6 x 6 mm3 Gaussian kernel. The general linear model (GLM) first level
analysis was conducted. BOLD responses were mapped as T-value maps overlaid onto our-in-house
sheep atlas template (Ella et al., 2017). Significance of BOLD responses was evaluated at cluster level
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using FDR -corrected p-values set to 0.01. Second level SPM analysis was conducted to compare
hypercapnia to baseline at each time point during LD and SD using a one sample t-test. A pvalue<0.05
was considered significant.
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DSC-MRI data were analysed using a Matlab toolbox adapted from Peruzzo et al. (2011) using
singular value decomposition (SVD) deconvolution (Zanderigo et al., 2009) and integrating both raw
and fitted arterial input functions (AIF) in the middle cerebral artery (MCA). The automatic algorithm
proposed by Peruzzo et al. (2011) was used for automatic selection of AIFs. Briefly, after drawing
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automatically a region of interest, the algorithm detected the MCA and a Γ-variate fit was computed
to remove the recirculation of contrast agent. A cluster analysis was performed to extract arterial
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voxels and the AIF was determined by averaging voxels in the responsive cluster.
2.2.3 Proton MR Spectroscopy
Proton MRS (1H-MRS) was conducted using a STEAM sequence (2048 points and a bandwidth of 3000
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Hz) and the following parameters (TR/TE/TM= 3000/5/30 ms; 256 acquisitions) in a large voxel of
interest (VOI = 10 x 12 x 13 mm3) covering the entire the hypothalamic structures (Fig. 7).
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FASTESTMAP (Gruetter R, 1993) was used for shimming down to a water linewidth of 10 ± 2Hz
within the hypothalamic structures. Both techniques came within the MRS package developed by
Edward J. Auerbach and Małgorzata Marjańska and provided by the Center for Magnetic Resonance
Research (CMRR) of the University of Minnesota under a C2P agreement. Collected MRS data were
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analyzed using LCModel (Provencher S, 1993) and metabolite quantification was obtained using the
unsuppressed water signal acquired in the same VOI (16 acquisitions). The water suppression was
performed using a VAriable Power and Optimized Relaxations delays (VAPOR) module (Tkac et al.,
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2021). Neurochemicals quantified with Cramér-Rao lower bounds (CRLB) to estimate the errors of
the neurochemical quantification under 30 % were considered reliable.
The basis set was provided by Stephen Provencher. It included simulated macromolecules and the
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following metabolites: alanine (Ala), Aspartate (Asp);Cr (Creatine); γ-amino butyric acid (GABA),
Glucose (Glc), Phosphocholine (PCho), Glycerophosphocholine (GPC), Glutamate (Glu), Glutamine
(Gln), N-Acetyl-Aspartate (NAA), N-Acetylaspartylglutamic acid (NAAG), Glucose (Glc), Lactate (Lac),
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Scyllo-Inositol (Scyllo), Taurine (Tau), myo-inositol (mI). Moreover the following sums were also
outputs of LCModel: total NAA (tNAA = NAA +NAAG), Glutamine + Glutamate (Glx) and total choline
(tCho).
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2.2.4 MRS voxel tracking and tissue segmentation:
The MRS VOI position was tracked using 2D T1-weighted Spin –Echo images (TR/TE=500/8.4 ms;
FOV=220 x 200 mm2; Matrix=128 x 128) acquired prior to MRS in the sagittal, coronal and axial
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planes. These images helped us for the accurate and identical positioning of the VOI for each ewe.
MP2RAGE images acquired in each ewe during both LD and SD periods were segmented using
Diffeomorphic Anatomical Registration Through Exponentiated Lie Algebra (DARTEL) (Ashburner
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J. 2007) and our in-house made anatomical sheep template (Ella et al., 2017). Tissue composition
inside the VOI was calculated based on the segmentation of 3D MPRAGE images using Gannet 3.0
(Harris et al., 2015). Water concentrations, used in LCModel analysis, were calculated based on the
volume fractions of white matter (WM), grey matter (GM) and cerebrospinal fluid (CSF), assuming
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water concentrations of WM, GM and CSF of 35,880, 43,300 and 55,556 mM, respectively.
Metabolite concentrations were then divided by the fraction of WM and GM to correct for CSF inside
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the VOI, since metabolites are mainly present in WM and GM (Near et al., 2021). The signal-to-noise
ratio (SNR) was obtained using N-acetylaspartate (NAA) peak height at 2.01 ppm divided by standard
deviation (SD) of noise collected between 8 and 10 ppm. For all spectra, LCModel quantification was
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performed on a spectral window between 0.2 and 4.2 ppm. Mean SNR values were 27 ± 9 in
hypothalamic structures. The water linewidth was 10 ± 2 Hz on average and the linewidth of total
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creatine was 11± 2 Hz for spectra acquired during LD and 9 ± 2 Hz for spectra acquired during SD with
no significant difference (p>0.05).
2.3 Statistical Analysis

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Data are presented as mean ± standard deviation unless otherwise stated and were analyzed using
the analysis of variance (ANOVA) for all metabolites together (repeated measures) and for rCBV
findings followed by Bonferroni tests for post-hoc comparisons. The factors analyzed were
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photoperiod (LD and SD) and Time (T01 and T02) during each period. Significance was considered for
p < 0.05. Normality tests were performed on data sets using a Kolmogorov-Smirnov test. Statistical
analyses were performed using GraphPad (Prism, San Diego, CA, USA).
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3. RESULTS:
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3.1 BOLD-fMRI within the hypothalamic structures
The BOLD fMRI responses to hypercapnia were compared during LD and SD. To ensure the specificity
of BOLD responses within hypothalamic structures, the BOLD responses to hypercapnia of different
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regions of the ewe brain were also compared during LD and SD periods. Individual BOLD responses to
hypercapnia Fig 3a and b) and group averaged BOLD T maps were compared (Fig 3 c and d) at two
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time points during LD and SD respectively. Significant and positive clusters of BOLD responses to the
hypercapnic challenge were detected over the whole brain during LD. Hypercapnia also activated the
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hypothalamus (Fig.3c, K=218, white arrow) at T01. During SD, BOLD responses to hypercapnia
remained positive at T01 except for hypothalamic regions, which showed negative clusters (Fig 3 d,
K= 21, white arrows). At T02, posterior brain areas responded positively whereas frontal,
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somatosensory and thalamic areas responded negatively (Fig 3e and f).
Two-sample t-tests were performed to compare LD and SD periods showing mainly positive changes
for the overall brain between LD and SD (Fig.4). Comparisons between LD2 and SD1 revealed
negative clusters within hypothalamic regions (arrows) (Fig. 4. B). Two-sample t-tests were also
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performed between BOLD T maps acquired at SD1 and SD2 (Fig. 4.D). Interestingly negative changes
were depicted, covering regions of the brain going from the cortex to the thalamus and surrounding
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the lateral ventricles. Positive clusters were depicted in the cerebral peduncles and within the
hypothalamus.
BOLD time courses were also obtained for the whole brain and for the hypothalamus (Fig.5.A-B and
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Fig. 5.E-F, respectively). Interestingly, the amplitudes of BOLD responses for the whole brain (WB)
were significantly higher during SD than LD (2.9 ± 0.8 % versus 1.6 ± 0.5 %, paired t test p=0.036, Fig.
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5C) whereas the amplitudes of the BOLD responses were significantly smaller during SD within the
hypothalamus (0.098±1.6% versus 2.7 ± 0.5% during LD; paired t test p=0.025). Within the
hypothalamus, negative BOLD responses were measured at the beginning of the SD period (-1.2± 0.6
%, SD1) (Fig. 5.D). BOLD responses became positive again towards the end of November (SD2, 1.4 ±
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0.5 %, SD2). During LD BOLD responses within the hypothalamus remained positive and constant
throughout this period (~ 2.7 ± 0.5 %, paired t test p>0.05) (Fig. 5.D).
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3.2 Cerebral Blood volume and Cerebral Blood Flow within the hypothalamus
rCBV and rCBF maps were obtained on a voxel by voxel basis during LD and SD (Fig.6 A and B).
Regions of interest were drawn within the hypothalamus. rCBV values increased significantly towards
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the second period of SD (SD2) relative to rCBV values obtained during LD (One way ANOVA, Dunn’s
post hoc test LD2 vs SD2 p<0.05, Fig. 6 C). During LD, rCBV remained constant within the
hypothalamus despite a non-significant rCBV drop during LD2-T02. No significant changes in CBF
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were measured between LD and SD and during each period.
3.3 Neurochemical profiling within hypothalamic structures

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Figure 7 depicts T1-weighted MPRAGE images with the position of the VOI within the hypothalamic
structures in the coronal (A), sagittal (B) and axial (C) planes. Representative examples of raw labeled
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proton MR spectra were also acquired within these VOIs during LD (D) and SD (E) and adjusted with
LCModel. The CRLBs are reported in Table 1.
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The neurochemical profiles within the hypothalamic structures acquired during LD and SD were
compared (Fig. 8.A, metabolite concentrations ± SD). A significant effect of Time was found (F (3,
264) =15.64; p< 0.0001) as well as a significant effect of the photoperiod (or the comparison between
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LD and SD) (F (1, 288) = 15.79; p<0.0001). The comparison between neurochemical profiles between
LD and SD yielded a significant overall increase in concentration for tNAA and Glx during SD relative
to LD (Bonferroni, t = 3.351(NAA) and t= 2.767 (Glx), p<0.05) (Fig.8.C, F). Between LD1 (T01) and LD2
(T02), mI (Fig. 8.D), NAA and tNAA concentrations decreased significantly (p<0.01, p<0.01 and
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p<0.001 respectively). There were no significant difference between neurochemical profiles between
LD1 and SD1 but a significant increase in Glu concentrations (p<0.05) and a significant decrease in mI
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concentration (p<0.05) between LD1 (T01) and SD2 (T02) while NAA and tNAA (p<0.05) increased
significantly between LD2 (T02) and SD1 (T01). Significant increases in Glu and NAA concentrations as
well as tNAA and Glx concentrations were also found (p< 0.001) during SD2 (T02) relative to LD2
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(T02). Finally, strong Glx concentration increases were revealed between SD1 (T01) and SD2 (T02)
(p<0.01).
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3.4 Relationships between vascular and metabolic changes.
No correlations were found between BOLD and metabolic concentration changes. Mean rCBV and
mean tNAA concentrations changes correlated strongly and showed similar evolutions across
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photoperiod (Fig.8G).
4. DISCUSSION:
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In the present paper, we proposed an early exploration of the vascular and neurochemical changes
induced by photoperiod in the sheep hypothalamic structures. We conducted BOLD fMRI, perfusion-
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MRI and in-vivo 1H MRS at 3T in the sheep brain under similar conditions to human scanning sessions.
To the best of our knowledge, this work represents the first in-vivo characterization of the vascular
and neurochemical consequences of photoperiodism using a combination of non-invasive MR
methods in the sheep brain. For an improved characterization of the metabolic changes, 1H-MRS
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measurements were performed longitudinally between April and July during LD and between
September and November during SD. The time points chosen during LD and SD correspond to the
basal level and the peak of AN (Batailler et al. 2018), occurring at the mid-time of their sexual rest
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period and sexual activity period respectively for the IF breed (Chanvallon et al., 2011).
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In the present study, hypercapnia induced significant BOLD changes within the brain of ewes as
expected. However, these changes varied according to the photoperiod and were induced over the
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whole brain. Within hypothalamic regions, hypercapnia induced negative BOLD responses at the
beginning of the SD period, which evolved into positive BOLD responses towards the end of the SD
period. Interestingly, negative BOLD responses to hypercapnia were revealed in other parts of the
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ewe brain during this same period. An Increase of the blood concentration in CO2 usually referred to
as cerebrovascular reactivity (CVR), represents the ability of blood vessels to dilate. In general, the
BOLD signal increases due to the dilation of blood vessels with CO2. However, negative CVR has also
been reported (Thomas et al., 2013). Negative BOLD responses to hypercapnia were attributed to a
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steal phenomenon as a result of a redistribution of blood flow from regions with exhausted
cerebrovascular reserve to areas with persevered vasodilatory capacity (Thomas et al., 2013;
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Poublanc et al., 2013). The steal effect has mainly been described for cerebrovascular diseases such
as Moyamoya disease or steno-occlusive diseases (Conklin et al., 2010). It has also been observed in
healthy subjects and CSF-rich regions within the healthy brain (Thomas et al., 2013). Since the goal
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of the present study is to characterize changes due to the photoperiod in regions of the ewe brain
close to the third ventricle and suspected to undergo neurogenic processes, the steal phenomenon
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might explain the negative CVR effects measured at the beginning of the SD period (SD1). Thomas et
al.(2013) attributed negative BOLD responses to hypercapnia to CBV effects rather than oxygenation
effects. However, hypoxia is involved in the regulation of stem cells where it can promote the
proliferation, migration and maturation of neural stem cells in both the SVZ and the dentate gyrus
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neurogenic niches (Gaifen et al., 2021). The existence of hypothalamic seasonal-dependent changes
within the ewe brain has been demonstrated in multiple studies (Migaud et al., 2011, 2015; Batailler
et al., 2017; Butruille et al., 2017). Moreover, our group investigated similar cohorts of ewes to the
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ones studied here and showed that these plastic processes occur primarily in the periventricular
space, which hosts seasonally regulated AN with higher neurogenic activity during SD. Our group
revealed that hypothalamic nuclei such as the arcuate nuclus of the hypothalamus (ARH) and the
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median eminence (ME) responded differentially to photoperiodism (Chevillard et al., 2022, 2023). In
particular, some nuclei showed increased vessel permeability whereas others showed increased
vascular density, which occurred during LD in the ARH. The Hypoxia -inducible factor 1α (HIF), a
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transcription factor that allows stem cells to adapt to hypoxic environments, did not reveal a
sensitivity to the photoperiod (Chevillard et al., 2022). In the present study, at SD1, both tNAA
concentrations and rCBV were significantly increased relative to LD2 but BOLD responses to
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hypercapnia were negative. As mentioned earlier, we suspect a vascular steal phenomenon close to
the third ventricle, which could also involve episodes of hypoxia promoting AN within the
hypothalamus in association with a reduced CVR and high metabolic activity (increased rCBV and
13
tNAA concentrations) at the beginning of the SD period (SD1). In cell cultures, lowered O2 promoted
the proliferation and differentiation of neural stem cells. In-vivo, cerebral ischemia promoted the
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generation of new neurons in the cerebral cortex (Gu et al., 2000) while neurogenesis and
angiogenesis were clearly linked in various stroke model studies (Ma et al., 2021). In addition,
intermittent hypoxia promotes proliferation of neural stem cells in SVZ and DG (Zhu et al, 2005). In
iew
the present study, the negative BOLD signals seen at SD1 in hypothalamic regions could represent
oxygenation changes to promote neural stem cell proliferation and differentiation, which could
explain the subsequent rise in rCBV and BOLD signals seen at SD2.
Within the sheep hypothalamus, the levels of Glx, and tNAA changed significantly between LD and
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SD. While mean NAA and tNAA changes were strongly correlated with mean rCBV changes at all time
points, significant increases in Glu and Glx concentrations took place at SD2-T02 together with
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significant increases in rCBV and BOLD responses relative to LD2-T02. This information suggests
increased vascular density, oxygenation, neuronal density and glutamate release supporting
neurotransmission at SD2-T02. er
The spatial resolution of our BOLD-fMRI and rCBV maps did not allow an accurate delineation of the
individual hypothalamic nuclei and a comparison of BOLD-fMRI and rCBV changes in the ARH and
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ME. Nevertheless, the present study suggests that both the vascular and oxygenation functions
undergo photoperiodic regulation in these regions.
LD corresponds to a period of food abundance for sheep outside in the fields at this time of the year.
During LD, a continuous decrease of mI, NAA and tNAA concentrations was observed. Previous
ot
studies in rodents have shown that lower levels of NAA correlated with higher levels of dopamine
release (Moffet et al., 2007, Ariyannur et al., 2013). Dopamine is considered a neurotransmitter
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involved in the motivational component of the reward-motivated behaviour. The period of food
abundance during LD would match with increasing levels of dopamine in the sheep brain. Moreover,
dopaminergic neurons have been implicated in the transition from the breeding period to the
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anestrous period and vice-versa, involving an important remodelling of the hypothalamic circuitry
(Lehman et al., 2010), which was found intriguingly more important during the anestrous period. Our
immunohistochemical findings in the ARH of the hypothalamus from another cohort of ewes
(Chevillard et al., 2022) showed important angiogenic processes during LD, which would indeed have
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significant effects on the metabolism of the hypothalamus. The changes in rCBV were not significant
during LD but a small inflection was observed at LD2 with significantly smaller rCBV values than at
SD1-T01 and SD2-T02, also corresponding to an inflection in tNAA at LD2-T02. NAA is a marker of
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neuronal density and its decrease in correlation with a decrease in rCBV supports both vascular and
14
metabolic remodeling processes within the hypothalamus prior to the onset of the sexual activity
period.
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A significant decrease of mI concentrations was also demonstrated between LD1 (T01) and LD2 (T02)
whereas levels were equivalent at subsequent time points. Moreover, mI concentrations during SD
were significantly correlated to progesterone concentrations (Fig.S2B). mI concentrations are usually
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higher in astrocytes than in neurons and apart from being an important organic osmolyte, mI is also
an intracellular messenger involved in transferring hormonal information. This role could explain the
correlation pattern between mI and progesterone concentrations seen in the present study.
Significant decreases in mI concentrations were also previously related to a volume-regulatory
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strategy of edema or to cell lysis and death further supporting our earlier hypothesis of cell death at
T01 during SD (Zhao et al., 2003; Harris et al., 2015).
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Despite this first in-vivo characterization of the vascular and neurochemical consequences of
photoperiod influence, our study has certain limitations. The ewes used in the present study were
healthy and underwent MRI and MRS under identical conditions during 2 periods of the year.
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However, for ethical and practical reasons, it was not possible to scan a larger cohort of ewes,
because 1) these animals weighed between 80 and 92 kg at the time of scanning and could not be
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manipulated like small animal models such as rodents, and 2) procedures had to include two hours
for full recovery after anesthesia.
Although sheep could be a valid model for translational studies (Murray et al., 2022), little is known
about their brain physiology and its characterization by MRI and MRS techniques. While recent
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consensuses on MRS techniques (Oz et al., 2014, Wilson et al., 2019) stated the requirements for
appropriate MRS acquisition in the human brain that are mostly applicable to the sheep’s
gyrencephalic brain, more investigations are necessary for an improved assessment of metabolic
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profiles in these animals under anesthesia. As ruminants, they also process and metabolize foods in a
different manner, which might also induce additional motion to the brain. Prior to surgery, it is highly
recommended to fast the animals for 24 hours and rumenostomy may be required.
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All the animals were intubated and anesthetized with isoflurane (1-3% in air). We did not observe an
impact of anesthesia on our data. However, the impact of anesthesia on adult neurogenesis was
investigated by others (Kim et al., 2020) lending no effects of isoflurane but the question remains
ep
highly debated (Changlian et al.2010).

Since increased AN was repeatedly observed within the hypothalamic structures of the sheep during
SD (Batailler et al., 2016), our results were interpreted assuming a simultaneous development of new
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cells in this structure. Immunohistochemical data acquired in the ARH of a different cohort of ewes in
our laboratory demonstrated significant neuronal and vascular changes in ARH and ME during LD and
SD (Chevillard et al., 2022; see supplementary section) However, large VOIs were used for MRS
15
measurements although the neurogenic niche is constrained to the ARH and ME within the
hypothalamus. Thus, metabolic observations were averaged over non-neurogenic and neurogenic
ed
tissues and cannot be considered entirely AN-dependent.). Increased spatial resolutions are
therefore needed for future investigations and increased specificity.
Here, we used a short echo STEAM sequence to acquire 1H MRS in hypothalamic structures at 3T. For
iew
an increased accuracy and reproducibility of metabolite quantification in such a deep brain structure,
high SNR is needed. In humans, increasing the magnetic field strength from 1.5T to 3T enhanced
significantly the quality of MR spectra acquired within the hypothalamus (Joers et al., 2017).
Moreover, the use of improved techniques such as short-echo semi- LASER sequences (Deelchand et
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al., 2021) enabled the longitudinal follow up of hyperglycemic patients and the preliminary
assessment of a linear correlation between hypothalamus and blood glucose concentrations (Joers et
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al., 2017). MRS studies in rodents also demonstrated improved results at higher field strength (14.1
T) with more sensitive sequences enabling the assessment of alterations of neurochemical changes in
the hypothalamus of high-fat fed and anorectic rodents (Lizarbe et al., 2019; Just et al., 2011a; Just et
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al., 2011b). In addition, the processing of MR spectra in the sheep brain could be improved with a
better tracking of motion, the measurement of sheep basis sets and of macromolecule spectra as
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advised in recent consensus papers (Oz et al., 2014, Wilson et al., 2019). Water concentrations, used
in LCModel analysis, were calculated assuming the volume fractions of WM, GM and CSF for the
human brain since no measurements of water content in the sheep brain were available.
Furthermore, the role of CSF in the brain and more specifically in the hypothalamus is gradually
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unfolding and appears significant (Zappaterra et al., 2012) but remains difficult to understand.
Notably, tanycytes, which are specialized glial cells lining the wall of the third ventricle are involved in
energy homeostasis, food intake and reproduction by regulating the blood brain barrier and the
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blood-CSF exchanges and modulating hypothalamic neurogenesis. These cells are also affected by
seasonality changes (Dardente H and Migaud M., 2021). Recent studies have shown that cilia, coating
some of the cells lining the wall of the 3rd ventricle drive CSF flow and might have significant effects
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on the directional CSF flow in the adjacent hypothalamus parenchyma (Eichele et al., 2020). In
particular, cilia-driven streams of signaling molecules offer an interesting perspective to understand
better the underpinnings of CSF-borne signals dynamically transmitted to the brain. Cerebrospinal
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fluid (CSF)-filled ventricles resemble dynamic reservoirs of signaling substances and nutrients that
can reach neurons, glia cells and stem cells within the hypothalamus. However, CSF does not flood
the hypothalamus but rather delivers important factors to specific hypothalamic nuclei, which
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function relate to metabolism, circadian timing or the control of stem cell proliferation and
differentiation. There is a concept of “volume transmission” (Agnati LF and Fuxe K., 2014), which
could explain how solutes and signaling molecules are transported. Therefore the tracking of changes
16
in cerebrospinal fluid partial volume (Joers et al., 2017) may improve outcomes since CSF exchange
bears important implications for a correct interpretation of photoperiod and AN-induced metabolic
ed
changes.
5. Conclusion:
Metabolic concentrations and vascular changes were determined for the first time using
iew
multiparametric MR data in the sheep hypothalamic structures at two opposite time points of the year
in terms of day length. The present study provides a framework for hypothesis testing on the impact of
metabolism and function on the physiological role of cellular plasticity in the adult brain. Investigations
on seasonal rhythms are relevant for a better understanding of the alterations of the hypothalamic-
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pituitary-gonadal axis during appetite and reproductive disorders potentially associated with changes
re
in AN activity levels.
Additionally, with a gyrencephalic brain and the possibility of being imaged under identical
conditions as humans in clinical scanners, the sheep represents a promising animal model for further
er
non-invasive imaging of AN and translational purposes (Just et al., 2022).
6. Conflict of Interest:
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Authors have no conflict of interest to disclose
7. Author contributions:
NJ and MM designed the study. NJ conducted the MR experiments. NJ, MM, PMC analyzed and
interpreted the data. MB and JPD contributed to animal experiments. MM, PMC and MB, PV and DP
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performed and analyzed immuno-histochemical data. PMC and MM wrote parts corresponding to
immunohistochemistry. NJ, PMC and MM wrote the manuscript. All authors read, corrected and
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approved the final version of the manuscript.

All MR related experiments were performed within the PIXANIM imaging platform of INRAE Nouzilly.
We are grateful for the preparation of animals, for the precious help from all the technical staff of
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the platform and for granting access to the platform.
8. Acknowledgements: This study was funded by a grant from Agence Nationale de la Recherche
(ANR-16-CE37-0006-01) to Martine Migaud. Nathalie Just received research funding from the
ep
Lundbeck Foundation (Experiment grant, grant nr. R370-2021-402)

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17
Table 1. Cramer-Rao Lower Bounds (CRLBs) ± standard deviation (sd)
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T01-LD1 ±sd T01-SD1 ±sd T02-LD2 ±sd T02-SD2 ±sd
Cr 6.75% 0.008 7.33% 0.005 9.00% 0.028 6% 0.01
Gln 25.50% 0.068 38.67% 0.187 31.00% 0.099 29% 0.05
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Glu 20.75% 0.068 18.33% 0.025 14.00% 0.014 15% 0.02
GPC 4.25% 0.004 8.67% 0.045 5.00% 0.014 8% 0.07
mI 5.00% 0.007 6.00% 0.000 6.00% 0.014 5% 0.01
NAA 13.00% 0.043 13.67% 0.053 9.00% 0.028 8% 0.02
GPC+PCho 4.25% 0.004 5.33% 0.005 5.00% 0.014 4% 0.00
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NAA+NAAG 7.00% 0.016 8.33% 0.012 9.00% 0.028 6% 0.01
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Glu+Gln 13.25% 0.038 14.33% 0.033 11.50% 0.021 11% 0.01
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ep
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18
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Figure Captions :
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Figure 1: Hypothalamic structures in gyrencephalic brains. A. Examples of labeled coronal section of

the sheep brain (Sheep Ovis aries) to illustrate hypothalamic structures surrounding the third
ventricle (from https://brains.anatomy.msu.edu/brains/sheep/scans/0200/image1.html and National
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Science Foundation [with granted permission from authors]). B. Sagittal and Coronal MRI images of
the sheep brain atlas (from Ella et al., 2017) (Th:Thalamus; Pit:Pituitary; Cx: Cortex; Cb: Cerebellum;
CC: Corpus Callosum; Hyp: hypothalamus; The yellow crossbars indicate the position of the
ep
hypothalamus) and coronal section with labels to illustrate hypothalamic structures in the sheep
brain surrounding the 3rd ventricle and paralleling human sections in Fig S1 (from
https://brains.anatomy.msu.edu/brains/sheep/scans/0200/image1.html and National Science
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Foundation [with granted permission from authors]).
23
Figure 2: Timeline and Protocols: Photoperiod describes the sensitivity of the sheep brain to the
ratio of day to night length. The sheep were scanned from May till July during long days (LD, lambing
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and beginning of anestrous period) and from late September till November during short Days (SD),
which also corresponds to the period of sexual activity in sheep (breeding period) and the period of
increased AN. During LD and SD, MRI and MRS took place twice during LD and twice during SD.
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During SD, blood samples were drawn to determine the physiological status of the ewes (luteal or
follicular phase of the cycle). 8 ewes were scanned per time point (T01 and T02) during each period.
Female sheep underwent BOLD fMRI under hypercapnic challenge, perfusion-MRI and 1H-MRS at 3T
for an enhanced sensitivity of structural, functional and metabolic parameters to the photoperiod
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and the adult neurogenesis (AN) within the hypothalamus structures.
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Figure 3: BOLD fMRI. a.b. Representative individual BOLD T maps at T01 and T02 during LD and SD.
c.d e.f. Group averaged BOLD T maps (n=8) obtained with one-sample t-tests (SPM) comparing
baseline and hypercapnic periods at T01 and T02 during LD and SD.
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Figure 4: BOLD effect comparisons between LD and SD. A. T maps comparing changes between LD1
and SD1 at T01. B. between LD1 at T01 and SD2 at T02 and C.between LD2 and SD2 at T02 ( 2-sample
t-test, SPM). D. T maps comparing SD at T01 and T02.
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Figure 5: BOLD time courses A. BOLD time courses during the hypercapnic challenge for the whole
brain (WB) during LD and SD at T01 and T02 (±SD). B. Mean BOLD responses for the whole brain and
the hypothalamus were compared between LD and SD showing significant differences between LD
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and SD both for the whole brain and for the hypothalamus. The evolution of BOLD responses at T01
and T02 during LD and SD were different for the WB and the hypothalamus. Within the
hypothalamus a significant decrease of BOLD amplitudes was found at SD1 compared to LD2 and SD2
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(p<0.001).C. BOLD time courses within the hypothalamus at T01 and T02 during LD and SD (±SD).
Figure 6: Perfusion MRI. A.B Representative Examples of rCBV and rCBF maps of the ewe brain. C.
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rCBV values were significantly different between LD2 and SD2 (p<0.05, 2-way ANOVA)
Figure 7: Proton Magnetic Resonance Spectroscopy (1H-MRS) in the hypothalamus. A-C: MPRAGE
coronal, sagittal and axial T1-weighted images depicting the position of the VOI within the
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hypothalamus (VOI = 10 x 12 x 13 mm3) and D-E: the corresponding MR spectra acquired during LD
and SD
Figure 8. Metabolite concentrations during the photoperiod A. Neurochemical profiles (± standard

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deviations) of the hypothalamus at LD1 (T01), SD1 (T01), LD (T02) and SD2 (T02). B. Comparison of
NAA concentrations at LD1, LD2, SD1 and SD2. C. Comparison of tNAA concentrations at LD1, LD2,
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SD1 and SD2. D. Comparison of mI concentrations at LD1, LD2, SD1 and SD2. E. Comparison of Glu
concentrations at LD1, LD2, SD1 and SD2. F. Comparison of Glx concentrations at LD1, LD2, SD1 and
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SD2.* p<0.05; ** p< 0.01; *** p<0.001 G. Correlation between mean tNAA and mean rCBV
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Supplementary Work
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Immunohistochemistry
Animals
Île-de-France primiparous non-gestating ewes were used in this study (n=10, 1.75±0.25-year-
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old). Animals were kept in sheepfold, under natural photoperiods, fed daily with alfalfa, corn, straw
and a supplement of vitamins and minerals and had ad libitum water. Animals were handled and
cared for in accordance with the EC Directive of 24 November 1986 (86/609/EEC) and with
authorization A37110 from the French Ministry of Agriculture.
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Tissue preparation
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Five brains were collected in October and five in May corresponding respectively to the short
photoperiod (Short Days/SD) and the long photoperiod (Long Days/LD). Prior to fixation, brains were
perfused through the carotid arteries with 1% sodium nitrite saline solution at 37°C (2l, 0.9% NaCl) to
wash the vascular bed. Brains were then fixed with 4% paraformaldehyde solution at 4°C (0.1M
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phosphate buffer; pH 7.4). Hypothalami were dissected from the optic chiasma to the
premammillary recess from fixed tissues and post-fixed in the 4% paraformaldehyde fixative solution
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for 48h. Hypothalamic blocks were then immersed in a 20% sucrose solution at 4°C for
cryoprotection. Prior to microcutting, blocks were frozen by immersion into isopentane at -50°C.
Coronal sections (14µm thick) were collected from a distance of 2.5mm rostral to the premammillary
recess using a cryostat (Leica CM 3050 S). The sections were directly mounted on Superfrost Plus
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slides (Fisher Scientific, Illkirch, France) and stored at -80°C until used for immunohistochemistry.
Three sections of the ARH were selected for immunolabeling (Fig. S2A).
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Immunolabeling
For brain section immunolabeling, an unmasking step was included in which sections were
subjected to heat mediated antigen retrieval using citric acid (pH 6). Sections were warmed to 90°C
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in a microwave oven in citrate buffer (0.01M, pH 6.0). After cooling to room temperature, sections
were rinsed three times in TBS (0.01M, pH 7.4). For all antibodies, sections were blocked and
permeabilized with TBS supplemented with 0.3% Triton and 5% mare serum respectively for 30min.
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The sections were incubated with primary antibodies (Table S1) diluted in the same solution
overnight at 37°C, followed by secondary antibodies (Table S1 ) at 37°C, in the dark, for 2h.Then, the
sections were counterstained in a solution of Hoechst (1µg/1ml dilution) at room temperature for 2
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min. The sections were next coverslipped using Fluoromount (Southern Biotech). For all antibodies,
normal serum IgGs of appropriate species were used as negative controls.
26
Quantification
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The densities of neural stem cells [SOX2+] and astrocyte [SOX2+S100+] and oligodendrocyte
[SOX2+OLIG2+] progenitors in the ARH were estimated using a semi-automated analysis process,
running on ImageJ (version 1.52, NIH, US) and Imaris software (version 9.6.0, Bitplane, UK) . A first
step consisting in the application of a 3x3pixels median filter has been used to improve signal/noise
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ratio. Densities of astrocyte and oligodendrocyte progenitors were conducted within an area of
interest of 800x800µm that lined the 3rd ventricle, called periventricular space. [SOX2+],
[SOX2+S100+] and [SOX2+OLIG2+] cells were automatically detected, using the Imaris “spot”
function following the use an established grey level threshold and an estimated cell radius of 6µm.
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Semi-automated Hoechst-positive cell detections were used as controls.
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Image capture
All images for figure preparation were acquired using a microscope slide scanner (Zeiss, Axio.Scan.
Z1) with Zen 2.3 software (version 4.0.3, Carl Zeiss, Oberkochen, Germany). Images shown in the
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different figures were pseudocolored using Imaris (version 9.6.0, Bitplane, UK) and adjusted for
brightness and contrast.
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Statistical analysis
Due to the small sample size, data are presented as mean values ± Standard Error of the Mean (SEM).
Statistical analyses were performed with GraphPad Prism (version 8.4.3) software. The normality of
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data was tested (Shapiro tests). As the data from [SOX2+] cells did not pass the normality test,
comparison between seasons was performed by a Mann-Whitney test, unlike the data of
[SOX2+S100+] and [SOX2+OLIG2+] cells which pass the normality test and for which the comparison
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between seasons was performed by an unpaired t-test. Results are presented as barplots.
Differences were considered statistically significant for a p-value ≤ 0.05.
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Higher neural stem cells, glial and oligodendrocyte progenitor cell densities in the arcuate nucleus
(ARH) during SD.
To investigate whether metabolic changes that occurred according to the seasons in the
ep
hypothalamus may reflect variations in the density of cell populations, we analyzed neural stem cells
([SOX2+]), astrocyte ([SOX2+S100+]) and oligodendrocytes ([SOX2+OLIG2+]) progenitor densities in
the periventricular space of the ARH during SD and LD (Fig. S2). Using an immunohistological
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approach associated to a semi-automated detection (Fig. S2) we showed an increase in [SOX2+]

densities (n=10, Mann-Whitney), as well as in [SOX2+S100+] and [SOX2+OLIG2+] (n=10, unpaired t-
test) progenitor densities during SD compared with LD (Fig. S2B-C).
27
ed
Table S1: Primary and secondary antibodies used for immunohistochemistry
Antigen Antibody Supplier/code Dilution
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SOX2 Anti-SOX2 goat polyclonal R&D systems (US), #AF2018 1:500
OLIG2 Anti-Olig2 rabbit polyclonal Millipore (USA), #AB9610 1:500
S100 Anti-S100 rabbit polyclonal DAKO (USA), #Z0311 1:500
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Species Conjugated Supplier/code Dilution
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Donkey anti-goat IgG Alexa488 Molecular Probes (USA), #A21202 1:1000
Donkey anti mouse IgG Alexa488 Molecular Probes (USA), #A21202 1:1000
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Donkey anti rabbit IgG Alexa 488 Molecular Probes (USA), #A21206 1:1000
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Supplementary Figures and captions
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Figure S1. Hypothalamic structures in gyrencephalic brains. A.Labeled schematic drawings
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of the human hypothalamus
(https://brains.anatomy.msu.edu/brains/human/hypothalamus/index.html and National
Science Foundation [with granted permission from authors]).
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Figure S2. A. Cyclicity of progesterone levels during the period of sexual activity of the 8 ewes used
in the present study from September to November. Shaded dark gray areas represent the MR
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scanning periods. Progesterone concentrations with similar dynamics (with same cyclicity) were
averaged across ewes (G1 n=3 ewes, G2 n= 3 ewes and G3 n= 2 ewes) for an easier visualization. The
important criterion was cyclicity and not that cycles may be shifted from one ewe to another. B
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Progesterone levels were correlated to Glu and mI concentrations during SD. Only mI concentrations
were correlated to progesterone levels (r= 0.62; p=0.015).
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Figure S3: Higher densities of neural stem cells, glial and oligodendrocyte progenitor cells were
found in the ARH during SP. A: Processing workflow used for the analysis of ARH cell densities. A
median filter (3x3pixels) was applied on raw photomicrographs before semi-automatic detection of
the markers neural stem cells [SOX2+], astrocyte [SOX2+S100+] and oligodendrocyte [SOX2+OLIG2+]
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progenitors respectively, performed on Imaris software. In the example, [SOX2+] are pseudocolored
in green, while cell nuclei are stained with Hoechst in blue (left panel). The semi-automated
detection of [SOX2+] is represented by white dots (right panel). B: Photomicrographs showing ARH
neural stem cells, and astrocyte and oligodendrocyte progenitors during LD and SD. [SOX2+] labelings
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are in green. [S100+] and [OLIG2+] cells are in magenta. Nuclei are stained with Hoechst (blue). Grey
arrows show double labelled cells ([SOX2+S100+] and [SOX2+OLIG2+]). Scale bar: 50µm. C: Panels
from left to right show the mean number ± SEM of [SOX2+], [SOX2+S100+] and [SOX2+OLIG2+]
respectively, estimated per mm² in the ARH depending on seasons (n=5). Data are represented as
barplots. For [SOX2+] a Mann-Whitney two tailed non-parametric test was used: * : p≤ 0.001. For
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[SOX2+S100+] and [SOX2+OLIG2+] unpaired t-tests were used: ***p ≤0.0001; **** p ≤0.0001.
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30
Figure S3
A Raw Filter Neural ROI Statistical

microphotography application progenitor cell 800x800µm analysis
detection
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DAPI (nuclei) Detected [SOX2+] cells
SOX2 (NPCs) ROI
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ARH ARH
3V
200µm
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B Neural progenitor cells Astrocyte precursor Oligodendrocyte
[SOX2+] cells precursor cells
[SOX2+S100+] [SOX2+OLIG2+]
SOX2 er SOX2 SOX2
S100 OLIG2
SD
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LD
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10µm
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C
[SOX2+OLIG2+] cells per mm²
[SOX2+S100+] cells per mm²
3000 1000 500

✱
[SOX2+] cells per mm²
✱✱✱✱
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2000
✱✱✱
500 250
1000
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0 0 0
SD LD SD LD SD LD
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Figure 8
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Highlights

Abbreviated title: BOLD fMRI and MRS of the sheep

ORCID: Nathalie Just: https://orcid.org/0000-0002-9401-0669; Martine Migaud: 0000-0002-9496-

Danish Research Centre for Magnetic Resonance (DRCMR), section 714

Office phone: +45 38626205 Email:nathaliej@drcmr.dk

Keywords: Ewes, hypothalamus, 1H-MRS, Adult neurogenesis, photoperiod

AN: Adult Neurogenesis

MRI: Magnetic Resonance Imaging

NEX: Number of excitations

VASO: Vascular space Occupancy

which astrocytes, tanycytes and microglia (Garcia- Caceres et al., 2019).

a total acquisition time of 11 minutes.

2.2.4 MRS voxel tracking and tissue segmentation:

2.3 Statistical Analysis

3.1 BOLD-fMRI within the hypothalamic structures

were measured between LD and SD and during each period.

3.3 Neurochemical profiling within hypothalamic structures

highly debated (Changlian et al.2010).

approved the final version of the manuscript.

the platform and for granting access to the platform.

Lundbeck Foundation (Experiment grant, grant nr. R370-2021-402)

Res. 392, 745-761. doi: 10.1007/s00441-023-03745-x.

with steal phenomenon is associated with increased diffusion in white matter of

D.G., 2010. A molecular switch for photoperiod responsiveness in mammals. Curr.

Magn. Reson. Imaging 42, 1431– 1440.1

by MRI at 3T. J. Magn. Reson. Imaging. 45, 681-691. doi: 10.1002/jmri.25383.

dehydration-induced anorexia in rats at 14.1 T using manganese-enhanced MRI and

in neuronal circuits. Nat. Rev. Neurosci. 7, 179–93. doi: 10.1038/nrn1867.

486-96. doi: 10.1177/0748730411420062.

Acetylaspartate in the CNS: from neurodiagnostics to neurobiology. Prog. Neurobiol.

658-79. doi: 10.1148/radiol.13130531.

Neuroimage 83, 505-12. doi: 10.1016/j.neuroimage.2013.07.005.

Experts' consensus recommendations. NMR Biomed. 34, e4459.doi:

Figure 1: Hypothalamic structures in gyrencephalic brains. A. Examples of labeled coronal section of

Foundation [with granted permission from authors]).

Figure 8. Metabolite concentrations during the photoperiod A. Neurochemical profiles (± standard

approach associated to a semi-automated detection (Fig. S2) we showed an increase in [SOX2+]

Antigen Antibody Supplier/code Dilution

OLIG2 Anti-Olig2 rabbit polyclonal Millipore (USA), #AB9610 1:500

S100 Anti-S100 rabbit polyclonal DAKO (USA), #Z0311 1:500

A Raw Filter Neural ROI Statistical

3000 1000 500

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