Frédéric Lévy
email: frederic.levy@inra.fr.

M Batailler

M Meurisse

M Keller

C Moussu

K Poissenot

M Migaud

Differential effects of oxytocin on olfactory, hippocampal and hypothalamic neurogenesis in adult sheep

Keywords: cell proliferation, cell survival, dentate gyrus, doublecortin, olfactory bulb, social behavior, ventromedian hypothalamic nucleus

New neurons are continuously added in the dentate gyrus of the hippocampus, the olfactory bulb and the hypothalamus of mammalian brain. In sheep, while the control of adult neurogenesis by the social environment or the photoperiod has been the subject of several studies, its regulation by intrinsic factors, like hormones or neurotransmitters is less documented. We addressed this question by investigating the effects of central oxytocin administration on hippocampal, olfactory and hypothalamic neurogenesis. Endogenous markers, Ki67, Sox2 and DCX were used to assess cell proliferation, progenitor cells density and cell survival respectively in non-gestant ewes receiving a steroid treatment followed by intracerebroventricular injections of either oxytocin or saline. The results showed that oxytocin treatment significantly decreases the density of neuroblasts in the olfactory bulb, increases the density of neuroblasts in the ventromedian nucleus of the hypothalamus while no change is observed in both ventral and dorsal dentate gyrus. In addition, no change in the density of progenitor cells is found in the three neurogenic niches. These findings show for the first time that in females, oxytocin can regulate adult neurogenesis by acting on neuroblasts but not on progenitor cells and that this regulation is region specific.

Introduction

In most mammals, newborn neurons are continuously generated throughout life mainly in two structures of the brain, the dentate gyrus (DG) of the hippocampus and the olfactory bulb [OB; [START_REF] Brus | Dynamics of olfactory and hippocampal neurogenesis in adult sheep[END_REF][START_REF] Curtis | Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension[END_REF][START_REF] Ming | Adult neurogenesis in the mammalian central nervous system[END_REF]. In the DG, stem cells reside in the subgranular zone and give rise to neuroblasts which become new granular neurons in the overlying granule cell layer [START_REF] Ming | Adult neurogenesis in the mammalian central nervous system[END_REF]. The neural stem cells located in the wall of the lateral ventricles constituting the subventricular zone (SVZ) produce transient amplifying cells, which divide to produce neuroblasts that, in turn, migrate toward the OB along the rostral migratory stream. After reaching the OB, the new cells mature into granular interneurons for the majority of them [START_REF] Lledo | Adult neurogenesis and functional plasticity in neuronal circuits[END_REF]. A neurogenic activity is also reported within the hypothalamus of several mammalian species [START_REF] Kokoeva | Evidence for constitutive neural cell proliferation in the adult murine hypothalamus[END_REF][START_REF] Kokoeva | Neurogenesis in the hypothalamus of adult mice: potential role in energy balance[END_REF][START_REF] Migaud | Seasonal changes in cell proliferation in the adult sheep brain and pars tuberalis[END_REF][START_REF] Migaud | Emerging new sites for adult neurogenesis in the mammalian brain: a comparative study between the hypothalamus and the classical neurogenic zones[END_REF].

Constitutive cell proliferation activity has been characterized in the arcuate and ventromedial nuclei and in the median eminence. Neural stem cells were detected among the cells lining the wall of the third ventricle and numerous immature neurons were found in the parenchyma of the ventromedial hypothalamus [VMH; [START_REF] Batailler | DCX-expressing cells in the vicinity of the hypothalamic neurogenic niche: a comparative study between mouse, sheep, and human tissues[END_REF][START_REF] Lee | Functional implications of hypothalamic neurogenesis in the adult mammalian brain[END_REF].

Extensive studies have shown that both extrinsic factors, like social environment or photoperiod, and intrinsic factors, like growth factors, neurotransmitters or physiological status and associated hormones, regulate each step of adult neurogenesis including proliferation and fate specification [for review : 32, 45, 51]. For instance, pregnancy and parturition are associated with dramatic hormonal changes and specifically in progesterone and estradiol plasmatic levels. In turn, steroid hormones, affect hippocampal, olfactory, and hypothalamic neurogenesis [for review : 3, 32, 51]. Some hormonal factors specifically regulate the activity of a neurogenic niche without affecting the other niches. For instance, in mice, prolactin increases cell proliferation in the SVZ although it does not affect hippocampal neurogenesis [START_REF] Shingo | Pregnancy-Stimulated Neurogenesis in the Adult Female Forebrain Mediated by Prolactin[END_REF] and high levels of corticosterone down-regulates hippocampal cell proliferation while no regulation of olfactory neurogenesis by corticosteroids has been reported in rats [START_REF] Brummelte | Chronic high corticosterone reduces neurogenesis in the dentate gyrus of adult male and female rats[END_REF]. Regarding oxytocin (OT), while this hormone is capable of stimulating hippocampal neurogenesis in male rats [START_REF] Leuner | Oxytocin stimulates adult neurogenesis even under conditions of stress and elevated glucocorticoids[END_REF], no evidence for its implication in the regulation of olfactory or hypothalamic neurogenesis has been so far reported. Interestingly, OT neurons in the hypothalamic paraventricular nuclei project to various brain regions including the OB, the CA3 hippocampal pyramidal cells and the mediobasal hypothalamus [MBH; [START_REF] Kendrick | Neural control of maternal behaviour and olfactory recognition of offspring[END_REF][START_REF] Lin | Oxytocin stimulates hippocampal neurogenesis via oxytocin receptor expressed in CA3 pyramidal neurons[END_REF][START_REF] Sofroniew | Morphology of vasopressin and oxytocin neurones and their central and vascular projections[END_REF] and OT receptors are found in the DG, on the periglomerular and granular interneurons of the OB and the VMH, the latest being included in the MBH [START_REF] Broad | Previous maternal experience potentiates the effect of parturition on oxytocin receptor mRNA expression in the paraventricular nucleus[END_REF][START_REF] Tribollet | Oxytocin receptors in the central nervous system. Distribution, development, and species differences[END_REF]. In sheep, parturition increases the expression of OT mRNA receptors in the VMH [START_REF] Broad | Previous maternal experience potentiates the effect of parturition on oxytocin receptor mRNA expression in the paraventricular nucleus[END_REF] and the OT release is enhanced in the OB at parturition and in the MBH during mating [START_REF] Kendrick | The role of oxytocin release in the mediobasal hypothalamus of the sheep in relation to sexual female receptivity[END_REF][START_REF] Lévy | Oxytocin and vasopressin release in the olfactory bulb of parturient ewes: changes with maternal experience and effects on acetylcholine, gammaaminobutyric acid, glutamate and noradrenaline release[END_REF].

Cell proliferation and neuronal survival are reduced in the early post-partum period in both the SVZ/OB and the DG of sheep [START_REF] Brus | Evidence for cell proliferation in the sheep brain and its down-regulation by parturition and interactions with the young[END_REF][START_REF] Brus | Interactions with the young downregulate adult olfactory neurogenesis and enhance the maturation of olfactory neuroblasts in sheep mothers[END_REF]. Since the whole oxytocinergic system is activated at this period [START_REF] Kendrick | Neural control of maternal behaviour and olfactory recognition of offspring[END_REF] and can act on the olfactory, hippocampal and hypothalamic neurogenic niches, the present study was designed to assess whether OT alters adult neurogenesis in the three niches. To this end, cell proliferation and cell survival were measured in non-pregnant ewes receiving steroid treatment followed by intracerebroventricular injections of OT mimicking endocrine events occurring at parturition and enabling induction of maternal behavior [START_REF] Kendrick | Intracerebroventricular oxytocin stimulates maternal behaviour in the sheep[END_REF][START_REF] Keverne | Oxytocin facilitation of maternal behavior in sheep[END_REF].

2.

Material and methods rostrocaudal plane, 30mm; mediolateral plane, 1.9 ± 0.4 mm from the middle of the third ventricle; depth, 18.1 ± 1 mm from the cortex surface (Fig. 1A). A pilot study allowed us to confirm that these coordinates were appropriate to avoid tissue damage to the SVZ and the hippocampus. Stainless steel guide-tubes were fixed to the bone with acrylic cement closed with obturators. The entire assembly was then protected with a Teflon ring anchored to the skull with stainless steel screws and acrylic cement. Following surgery, animals received daily intramuscular injections of a solution containing an antibiotic (5 days, 5 ml of amoxicillin, Centravet, Plancoët, France), an anti-inflammatory (5 ml diurizone for 3 days, 2 ml finadyne on the first day, Centravet, Plancoët, France) and an analgesic (morphine (dose) for the first day, CDM Lavoisier).

Treatment

Two weeks after surgery, ewes were housed in 2×1 m individual pens and were daily accustomed to human presence and handling for a week (Fig. 1B). After receiving vaginal sponges impregnated with a progestagen for 14 days, ewes received two daily subcutaneous injections of estradiol benzoate (200µg /day). This steroid treatment was used because progesterone suppresses the basal release of OT sensitizing the brain to a surge of OT following estrogen injections and estradiol increases OT synthesis in the brain as well as the number of OT receptors [START_REF] Broad | Changes in oxytocin immunoreactivity and mRNA expression in the sheep brain during pregnancy, parturition and lactation and in response to oestrogen and progesterone[END_REF][START_REF] Caldwell | Estrogen increases affinity of oxytocin receptors in the medial preoptic areaanterior hypothalamus[END_REF].

The day following the steroid treatment and for two days, ewes were unilaterally Kendrick's study [START_REF] Keverne | Oxytocin facilitation of maternal behavior in sheep[END_REF] showing that central administration of OT in non-pregnant ewes induces the all repertoire of maternal behavior normally observed at parturition. We chose two daily OT i.c.v. injections as it was reported that repeated OT injections enhanced the number of adult-generated neurons [START_REF] Leuner | Oxytocin stimulates adult neurogenesis even under conditions of stress and elevated glucocorticoids[END_REF].

Brain perfusion and immunohistochemistry

Four hours after the end of the second intracerebroventricular infusion, ewes were deeply anesthetized with thiopental and decapitated by a licensed butcher.

Ewes were rapidly killed after the second injection since one of our aim was to investigate the effects of OT injection on cell proliferation. In the case of a longer time interval between injection and sacrifice, a causal link would have been difficult to establish.

The heads of the ewes were immediately perfused via the carotid arteries with 2 L of warm 1% sodium nitrite in phosphate buffer saline, followed by 4 L of ice-cold 4% paraformaldehyde solution in 0.1M phosphate buffer (pH 7.4). The brain was dissected out, cut into several blocks (MOB, dHipp, vHipp, SVZ and VMH) and postfixed in the same fixative for 48 h. The tissues were then stored in 20% sucrose for at least 2 days until sectioned. Frontal sections of the SVZ, the MOB, the dorsal and ventral hippocampus (dHipp and vHipp) and the VMH were cut on a cryostat at a thickness of 30μm. Sections were stored at 4 °C in TBS Azide buffer until their use for immunostaining. Examination of frontal sections at the level of the lateral ventricle confirmed that all the cannulae were localized within the lateral ventricle.

To reveal the putative effect of OT administration on cell proliferation in the 3 neurogenic niches, Ki-67, an endogenous marker of cell division expressed for the entire duration of the mitotic process [START_REF] Kee | The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis[END_REF], was used in the SVZ and the dHipp. In the VMH, Sox2, used as a marker for neural stem /progenitor cells [START_REF] Llorens-Martin | Multiple birthdating analyses in adult neurogenesis: a line-up of the usual suspects[END_REF], was used in 6 animals out of 8, since this area was irreversibly damaged in 2 animals during dissection [START_REF] Episkopou | SOX2 functions in adult neural stem cells[END_REF][START_REF] Ferri | Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain[END_REF][START_REF] Migaud | Emerging new sites for adult neurogenesis in the mammalian brain: a comparative study between the hypothalamus and the classical neurogenic zones[END_REF].

To reveal Ki67-positive cells a peroxidase single-immunolabeling was used.

Sections were mounted on a glass slide and dried at 37°C. Then the sections were warmed up to 90°C in citrate buffer 0.01M using a microwave. After cooling 1 h to room temperature and three rinses in TBS-Triton 0.3% (TBST), sections were preincubated for 10 min in TBST-0.1%-sodium azide-BSA 1% (TBSTA-BSA 1%), then incubated overnight at room temperature in primary mouse anti-Ki67 (1:250; BD Pharmingen, USA). The next day, sections were rinsed four times in TBST, and incubated at room temperature for 2.5 h with a sheep anti mouse peroxidase antibody (1:300; Jackson, USA). Finally, after two rinses in TBS and two rinses in Tris-HCl, sections were reacted for peroxidase detection in DAB-Ni-H2O2 that allows a permanent labeling of Ki67 positive cells appropriate for counting.

In the VMH, previous experiments showed that, Sox2 fluorescent immunolabeling was a relevant indicator for the neurogenic activity [START_REF] Batailler | Sensitivity to the photoperiod and potential migratory features of neuroblasts in the adult sheep hypothalamus[END_REF]. After 30mn in 0.1% sodium borohydride in TBS at room temperature followed by three rinses in TBS, the sections were incubated for 1 h in TBSTA-BSA 1% at room temperature.

They were then incubated for 48 h in goat anti-Sox2 (1:300; R and D Systems, USA).

They were rinsed four times for 5 min in TBST before incubation (for 3h at 4°C) in CY3-donkey anti goat (1:1000; Jackson, USA). After three rinses in TBST, cells nuclei were counterstained in a Hoechst solution diluted at 1:5000 in TBS, followed by three rinses of TBS. The immunostained sections were then mounted on slides, dried and immersed 10 min in 0.1% Sudan Black B (Merck, Germany) diluted in 70% ethanol. Finally, slides were washed two times in ethanol (5mn) and one time in TBS (5 min). They were then dried and coverslipped with Fluoromount-G (Southern Biotech, Birmingham, AL).

Doublecortin (DCX) is expressed in migrating neuroblasts [START_REF] Brown | Transient expression of doublecortin during adult neurogenesis[END_REF][START_REF] Gleeson | Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons[END_REF][START_REF] Nacher | Doublecortin expression in the adult rat telencephalon[END_REF] and is a reliable and specific marker that reflects levels of adult neurogenesis [START_REF] Brown | Transient expression of doublecortin during adult neurogenesis[END_REF][START_REF] Brus | Dynamics of olfactory and hippocampal neurogenesis in adult sheep[END_REF]. Young proliferating neuroblasts could also express DCX together with either Ki67 or Sox2

indicating cell division of neuroblasts [START_REF] Brown | Transient expression of doublecortin during adult neurogenesis[END_REF]. This marker was used to reveal the effect of OT administration on olfactory, hippocampal and hypothalamic neurogenesis.

For DCX immunolabeling, peroxidase technique was used for the OB, the dHipp, the vHipp. After two rinses in TBST and two rinses in Tris-HCl, sections were reacted in DAB-Ni-H2O2 allowing a permanent labeling of DCX positive cells appropriate for counting. The slides were then rinsed, dried, dehydrated through graded alcohol, cleared in toluene and cover-slipped with Depex®.

In the VMH, previous experiments showed that fluorescent immunolabeling of DCX+ cells provided a detailed distribution pattern of immature and developing neurons in sheep [START_REF] Brown | Transient expression of doublecortin during adult neurogenesis[END_REF][START_REF] Gleeson | Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons[END_REF][START_REF] Nacher | Doublecortin expression in the adult rat telencephalon[END_REF]. Before the antigen retrieval with a microwave method, sections were mounted on a glass slide and dried at 37°C. Then the sections were warmed up to 90°C in citrate buffer 0.01M using a microwave. After cooling to room temperature and three rinses in 0.3% TBST, sections were pre-incubated for 10 min in TBST-1% horse serum, and then incubated in primary goat anti-DCX (1:300, Santa Cruz Biotechnology, California, USA) in TBST-0.1% sodium azide-1% horse serum overnight at room temperature. The next day, sections were rinsed three times for 10 min in TBST, and incubated at room temperature for 3 h with a HRP-donkey anti-goat (1:300; Jackson, USA) for peroxidase labeling or with CY3-donkey anti-goat for immunofluorescence (1:1000; Jackson, USA).After three washes in TBST slides were immersed for 2 min in a Hoechst solution (1:5000; Molecular Probes/Invitrogen, Carlsbad, CA) for nuclear staining and rinsed in TBS. The slides were dried, immersed 10mn in 0.1% Sudan Black B (Merck, Germany) diluted in 70% ethanol and washed twice in ethanol (5 min) and once in TBS (5 min). They were then dried and coverslipped with Fluoromount-G (Southern Biotech, Birmingham, AL).

Quantification

The number of Ki67, Sox2 and DCX positive cells was assessed by counting the immunostained sections using a photonic microscope (Axioscop,Zeiss, Germany) with a motorized stage on a magnification of x20 and a cell-count analysis software (Mercator, Explora Nova, La Rochelle, France), according to a predetermined grey level and size criteria. Sections per animal were selected through different levels along the rostrocaudal axis of the structures: 3 sections for the OB, the SVZ, the vHipp and the dHipp and 4 sections every 500µm for the VMH. Each area of interest was drawn with a x2.5 magnification. In the OB, granular layer was drawn and randomly covered by probes (300 µmX200 µm) separated from each other by 250µm. Both in the dHipp and vHipp, 6 rectangles (300 µmX50 µm) were randomly distributed along the dentate gyrus. The density of immunostained cells was then calculated. In the VMH, counting was performed within 2 rectangles (800µmX1400µm) placed next to the fornix, one on the right side of the brain, one on the left side.

Because Ki67 and Sox2 can also be expressed by some young proliferating neuroblasts [START_REF] Brown | Transient expression of doublecortin during adult neurogenesis[END_REF], it is possible that counting of the population of proliferative cells in the three neurogenic niches involves some immature neurons.

Statistical analysis

Due to lack of normality, data were analyzed with non-parametric Mann-Whitney tests. Statistical analyses were performed using the statistical package

Statistica 10 and the level of statistical significance was set at p≤0.05. All data are represented as median and interquartile ranges.

Results

Proliferation

Representative cell labelings for Ki67 in the SVZ and in the dHipp are shown in Fig. 2A. Following immunolabeling quantification, Ki67 + cell density in the SVZ did not significantly differ between OT and control groups (Z=1.26, p=0.2; Fig. 2B).

Similarly, no significant difference was observed in the dHipp for Ki67 + cell density between OT and control group (Z=0.

Discussion

The present study shows for the first time in steroid-primed females that acute central administration of OT regulates adult neurogenesis in a region specific manner. OT treatment results in a decreased density of neuroblasts (DCX + cells) in the OB, an increased density of neuroblasts in the VMH while no change is found in both vHipp and dHipp. In addition, no change in cell proliferation (Ki67 + and Sox2 + cells) is observed in the three neurogenic niches.

Previous studies in sheep reported that, at parturition, an increased release of OT occurs within the granular layer of the OB [START_REF] Lévy | Oxytocin and vasopressin release in the olfactory bulb of parturient ewes: changes with maternal experience and effects on acetylcholine, gammaaminobutyric acid, glutamate and noradrenaline release[END_REF]. In addition, during the peripartum period, a down-regulation of the number of neuroblasts (DCX + cells) has been reported within the granular layer of the OB [START_REF] Brus | Interactions with the young downregulate adult olfactory neurogenesis and enhance the maturation of olfactory neuroblasts in sheep mothers[END_REF]. Thus, the present study suggests that OT can be responsible for this reduction in new olfactory neurons occurring at parturition. A considerable body of evidence reveals that OT is involved in various olfactory guided behaviors [START_REF] Grinevich | Interplay between Oxytocin and Sensory Systems in the Orchestration of Socio-Emotional Behaviors[END_REF][START_REF] Lieberwirth | Social bonding: regulation by neuropeptides[END_REF]. In the context of mother-young interactions, OT regulates preference of rat pup for maternal odors [START_REF] Nelson | Oxytocin mediates acquisition of maternally associated odor preferences in preweanling rat pups[END_REF] and OT administration within the OB induces maternal behavior in ovariectomized rats primed with estrogen [START_REF] Yu | The olfactory bulb: a critical site of action for oxytocin in the induction of maternal behaviour in the rat[END_REF].

In ewes, i.c.v. OT administration induces olfactory preference for amniotic fluid odors [START_REF] Lévy | Olfactory mediation of maternal behavior in selected mammalian species[END_REF][START_REF] Lévy | Physiological determinism of olfactory attraction for amniotic fluids in sheep[END_REF]. Thus, since olfactory newborn neurons are involved in lamb odor recognition [START_REF] Corona | Disruption of adult olfactory neurogenesis induces deficits in maternal behavior in sheep[END_REF][START_REF] Corona | Exposure to young preferentially activates adult-born neurons in the main olfactory bulb of sheep mothers[END_REF], it is possible that OT drives changes in odor preference by acting on olfactory neurogenesis. Interestingly, interactions with the lamb at parturition downregulate olfactory neurogenesis in sheep mothers favoring maturation of neuroblasts [START_REF] Brus | Interactions with the young downregulate adult olfactory neurogenesis and enhance the maturation of olfactory neuroblasts in sheep mothers[END_REF] and disruption of this neurogenesis prevents olfactory memory of the lamb [START_REF] Corona | Exposure to young preferentially activates adult-born neurons in the main olfactory bulb of sheep mothers[END_REF].

Therefore, OT by decreasing the density of neuroblasts could also participate in the formation of this olfactory memory. The effect of OT could be direct through the activation of specific receptors expressed by olfactory neuroblasts (DCX + cells) or by more mature adult-generated neurons. Alternatively, an indirect effect of OT through an intermediate neuronal population can be also considered. In rodents, neural progenitors in the DG do not express OT receptors and OT stimulates neurogenesis in the DG via oxytocin receptor expressed in CA3 neurons [START_REF] Lin | Oxytocin stimulates hippocampal neurogenesis via oxytocin receptor expressed in CA3 pyramidal neurons[END_REF]. The fact that OT can activate autoreceptors on OT neurons of the paraventricular nucleus of the hypothalamus [START_REF] Freund-Mercier | Oxytocin receptors on oxytocin neurones: histoautoradiographic detection in the lactating rat[END_REF] that provides afferent input to the OB [START_REF] Gimpl | The oxytocin receptor system: structure, function, and regulation[END_REF][START_REF] Lévy | Afferents to the rostral olfactory bulb in sheep with special emphasis on the cholinergic, noradrenergic and serotonergic connections[END_REF] could be a mechanism by which OT regulates olfactory neurogenesis.

Intracerebroventricular administration of OT has no influence on cell proliferation (Ki67 + cells) in the SVZ. Similarly, a lack of effect on cell proliferation in the SVZ of rats was reported after OT administration [START_REF] Leuner | Oxytocin stimulates adult neurogenesis even under conditions of stress and elevated glucocorticoids[END_REF]. This lack of OT effect could be due to the absence of OT receptors in the SVZ, although this hypothesis requires further analysis. Alternatively, OT may modulate cell proliferation indirectly by acting on OT-responsive neurons within regions providing afferents to the SVZ, as it was reported in the control of the SVZ neural stem cells by hypothalamic proopiomelanocortin neurons [START_REF] Paul | Hypothalamic regulation of regionally distinct adult neural stem cells and neurogenesis[END_REF]. According to this hypothesis, the amount of OT administered in the lateral ventricles would not have been sufficient to activate the OT-responsive neurons.

No change in either cell proliferation (Ki67 + cells) or in neuroblasts (DCX + cells) production in the DG is found after i.c.v. OT administration. In rodents, contradictory results were reported: an enhancement of cell proliferation in the vHipp of male rat, but not the dHipp, following peripheral or central OT administration [START_REF] Leuner | Oxytocin stimulates adult neurogenesis even under conditions of stress and elevated glucocorticoids[END_REF] whereas no change in progenitor cell proliferation and differentiation into immature neurons induced by conditional deletion of OT receptors in male mice [START_REF] Lin | Oxytocin stimulates hippocampal neurogenesis via oxytocin receptor expressed in CA3 pyramidal neurons[END_REF]. However, beside sex differences between rodent and sheep studies, OT receptor distribution varies between species [START_REF] Gimpl | The oxytocin receptor system: structure, function, and regulation[END_REF][START_REF] Tribollet | Oxytocin receptors in the central nervous system. Distribution, development, and species differences[END_REF]. For instance, OT binding sites are lacking in the hippocampus, amygdala, entorhinal cortex, and olfactory bulb of human brains [START_REF] Loup | Localization of highaffinity binding sites for oxytocin and vasopressin in the human brain. An autoradiographic study[END_REF].

In sheep, very low levels of OT receptor mRNA expression has been found in the DG and steroid treatment did not contribute to increase expression [START_REF] Broad | Previous maternal experience potentiates the effect of parturition on oxytocin receptor mRNA expression in the paraventricular nucleus[END_REF]. Consequently, regulation of hippocampal neurogenesis would not involve OT due to a paucity of OT receptors in the DG which is consistent with the inability of OT to stimulate neurogenesis in the dHipp of rats which contains low levels of OT receptors [START_REF] Liberzon | Effects of stress and glucocorticoids on CNS oxytocin receptor binding[END_REF]. It is possible that other neuromodulatory factors contributed to the regulation of hippocampal neurogenesis, such as glucocorticoids, which are potent inhibitors of neurogenesis in rodents [START_REF] Brummelte | Chronic high corticosterone reduces neurogenesis in the dentate gyrus of adult male and female rats[END_REF][START_REF] Mirescu | Stress and adult neurogenesis[END_REF][START_REF] Wong | The corticoid environment: a determining factor for neural progenitors' survival in the adult hippocampus[END_REF][START_REF] Wong | Raised circulating corticosterone inhibits neuronal differentiation of progenitor cells in the adult hippocampus[END_REF]. Alternatively, doses and timing of OT injections would not be appropriate. Considering the low level of OT receptors in the DG, the OT treatment would not be long enough to induce changes in hippocampal neurogenesis.

Central OT administration induces an increase in the number of neuroblasts (DCX + cells) in the VMH. It remains unclear whether the effects of OT are mediated via the activation of OT receptors on neuroblasts and it would be interested to examine whether hypothalamic neuroblasts express OT receptors. Nevertheless, the VMH of female sheep contains high levels of OT receptors and a steroid treatment greatly enhances OT receptor expression [START_REF] Broad | Changes in oxytocin immunoreactivity and mRNA expression in the sheep brain during pregnancy, parturition and lactation and in response to oestrogen and progesterone[END_REF]. Increased OT release within the MBH, which comprises the VMH, is reported in ewes during mating and OT infusion by retrodialysis in the MBH decreases sexual receptivity [START_REF] Kendrick | The role of oxytocin release in the mediobasal hypothalamus of the sheep in relation to sexual female receptivity[END_REF]. The up-regulation of neurogenesis induced by OT release during mating could provide a mechanism by which OT inhibits sexual receptivity. The mechanism could involve an effect of OT on VMH neuroblasts by modulating their dendrite length reaching the lateral fiber complex, those projections being involved in the control of female sexual behavior [START_REF] Griffin | Ovarian Hormone Action in the Hypothalamic Ventromedial Nucleus: Remodelling to Regulate Reproduction[END_REF]. In addition, VMH neuroblasts could migrate towards rostral parts of the hypothalamus, including the preoptic area involved in sexual behavior [START_REF] Batailler | Sensitivity to the photoperiod and potential migratory features of neuroblasts in the adult sheep hypothalamus[END_REF].

In conclusion, these findings constitute a first evidence for OT regulation of adult neurogenesis in female. This regulation could be involved in the major role played by OT in socio-sexual interactions [START_REF] Grinevich | Interplay between Oxytocin and Sensory Systems in the Orchestration of Socio-Emotional Behaviors[END_REF]. No significant difference was observed between OT and control groups in the three structures.

  infused with artificial cerebrospinal fluid (aCSF; pH 7.4; Control group, N= 8) or bilaterally with OT (OT group, N= 8; 25µg/per side/day) by inserting perfusion needles (Cooper Needles; UK) into the guide cannulae positioned into the lateral ventricle. One milliliter of solution was infused over a period of 60 minutes in one side for the CSF or both sides for OT. The OT dose was adapted fromKeverne and

  figures 3A and 3C. Quantification analysis reveals that in the granular layer of the

Figure 1 :

 1 Figure 1: Timetable and protocol design.

Figure 2 :

 2 Figure 2: Effects of OT i.c.v. infusion on cell proliferation in the SVZ, the dHipp and

Figure 3 :

 3 Figure 3: Effects of OT i.c.v. infusion on neurogenesis in the OB and the dHipp.

(

  A). Frontal section of the OB stained with Cresyl Violet (scale bar: 1mm) and immunostaining of DCX+ cells in the control and OT groups (scale bar: 50µm). VOB: ventricular olfactory bulb, Gl: glomerular cell layer, Gr: granular cell layer. (B). Results are presented as median and interquartiles of the number of DCX+ cells per mm 2 in the OB. OT infusion induces a significant decrease in the number of DCX+ cells per mm 2 . (C). Frontal section of the dHipp showing DCX immunolabeling in the dentate gyrus (scale bar: 25 µm) and higher magnification of DCX immunolabeling in the control and OT groups (scale bar: 500µm). DG: dentate gyrus. (D). Results are presented as median and interquartiles of the number of DCX+ cells per mm 2 in the dHipp. No significant difference was observed between OT and control groups.

Figure 4 :

 4 Figure 4: Effects of OT i.c.v. infusion on neurogenesis in the VMH.

Acknowledgments

The authors would like to acknowledge the financial support of the ANR programme blanc(2012-2015) PLASTMATBEHAV and the ANR-16-CE37-0006-01 HypNeurogen . We particularly thank (1) J.-P. Dubois for sacrifice of animals; (2) D.

Capo and its staff for animal breeding; (3) the cellular imaging platform (PIC) of UMR PRC, Nouzilly. BIBLIOGRAPHY


MD Marie Pouzoullic

MD, PhD Anne-Marie Schott

MD, PhD Dolores Sánchez-Rodríguez

MD, PhD Regis Bataille

MD, PhD Cedric Annweiler
email: cedric.annweiler@chu-angers.fr

MD Cédric Annweiler

The International St-aging System as a prognostic marker in general senior population: findings from the EPIDOS cohort study

Keywords: 2, Figure count: 2 albumin, beta-2 microglobulin, Multiple Myeloma International Staging System, prognosis, survival, older adults HIGHLIGHTS

The International Staging System(ISS) -calculated from serum albumin and beta-2 microglobulin(β2m)-is an established prognostic marker in multiple myeloma(MM), which has also been suggested to account for survival among general senior population. Our objective was to examine long-term survival of older women free of MM according to baseline ISS. The study included 230 community-dwelling healthy older women without known MM from the EPIDOS cohort(mean±SD, 80.4±3.4years). Serum albumin and β2m

were measured at baseline, and used to calculate the ISS a posteriori. Abnormal ISS was defined as ISS=2 or ISS=3, although ISS=1 was considered normal. The vital status was sought after a mean follow-up of 17.6±0.2years(range, 16.8-18.3). Age, body mass index, mean arterial pressure, diabetes mellitus, hypertension, coronary heart disease, stroke, use corticosteroids, number of drugs daily taken, smoking, physical activity, fall history, bone mineral density, and creatinine clearance were used as potential confounders. All participants died during the 17-year follow-up. Compared to women with normal ISS, those with abnormal ISS(n=24) had shorter survival time(4.9±4.3 versus 8.7±5.2years, P=0.001) and died earlier(85.6±4.8 versus 89.1±5.6years old, P=0.003). Survival time after blood test correlated with both serum albumin(r=0.16, P=0.015) and β2m(r=-0.27, P<0.001). Cox regression revealed that abnormal ISS was associated with mortality(adjusted HR=3.03, P<0.001). Kaplan-Meier distributions showed that participants with abnormal ISS died earlier than those with normal ISS(log-rank P<0.001). In conclusion, community-dwelling older women with abnormal ISS had shorter survival time than the others, suggesting that ISS could be considered as a universal prognostic "aging system" rather than a specific MM "staging system".

INTRODUCTION

The first step of any efficient geriatric care relies on the early identification of older adults most vulnerable to stress, who are at greater risk of failure of usual coping mechanisms, with consequent lower survival chance. This screening is yet limited by the difficulty of implementation into clinical practice due to the time consumption of standard validated tools [START_REF] Bergman | Frailty: an emerging research and clinical paradigm issues and controversies[END_REF][START_REF] Rodrigues-Manas | Searching for an operational definition of frailty: a Delphi method based concensus Statement. The Frailty Operativ Definition-Consensus Conference Project[END_REF]. A simple and easily accessible blood test is thus desirable to improve the feasibility of the screening for older adults with limited life expectancy in daily clinical practice.

There is ample evidence that serum albumin (SA) and beta-2 microglobulin (β2m) are both biological prognostic biomarkers of survival [START_REF] Goldwasser | Association of serum albumin and mortality risk[END_REF][START_REF] Corti | Serum albumin level and physical disability as predictors of mortality in older persons[END_REF][START_REF] Shinkai | Beta2-microglobulin for risk stratification of total mortality in the elderly population: comparison with cystatin C and C-reactive protein[END_REF]. The combination of SA and β2m constitutes a potent staging system in multiple myeloma (MM) [START_REF] Bataille | Prognostic factors and staging in multiple myeloma[END_REF][START_REF] Durie | A clinical staging system for muliple myeloma[END_REF][START_REF] Durie | Prognostic value of pre-treatment serum B2 microglobulin in myeloma: a Southwest Oncology Group study[END_REF][START_REF] Jacobson | A new staging system for multiple myeloma patients based on the Southwest Oncology Group (SWOG) experience[END_REF][START_REF] Ph | International staging system for Multiple Myeloma[END_REF], with a higher prognostic value compared to all other markers including gene expression profiles [START_REF] Jacobson | A new staging system for multiple myeloma patients based on the Southwest Oncology Group (SWOG) experience[END_REF]. In 2005, this SA/β2m combination was internationally recognized as the MM international staging system (ISS) [START_REF] Ph | International staging system for Multiple Myeloma[END_REF]. However and importantly, it was proposed that the ISS may actually take into account more the age-related comorbidity burden of MM older patients than the intrinsic malignancy of the MM clone [START_REF] Bataille | Multiple Myeloma International Staging System: "Staging" or Simply "Aging" System?[END_REF]. Decreased SA and increased β2m reflect declines across multiple physiologic systems and may account for the comorbidity burden among older adults [START_REF] Bataille | Multiple Myeloma International Staging System: "Staging" or Simply "Aging" System?[END_REF]. This suggests that the ISS should not be considered as a specific MM staging system, but rather as an "aging system", i.e. a universal prognostic biomarker in older adults.

We hypothesized that the ISS could predict survival time in general senior population. We had the opportunity to examine the association of ISS with survival time in a sample of healthy women aged ≥75years from the large representative community-based EPIDémiologie de l'OStéoporose (EPIDOS) cohort. The objective of this historical, longitudinal cohort study was to examine long-term survival of older women according to baseline ISS.

MATERIALS AND METHODS

We studied 230 participants without known MM, a subset recruited from 1992 to 1994 in the EPIDOS study, a French large observational prospective multicenter cohort study designed to evaluate risk factors for hip fractures among healthy community-dwelling women aged 75 years and older [START_REF] Dargent-Molina | Fall-related factors and risk of hip fracture: the EPIDOS prospective study[END_REF]. The present analysis considered data from all women who had: i) full data on serum measures; and ii) full data on mortality up to October 1, 2010.

All study participants had a blood test at the time of inclusion. Sera were stored at -100°C until analyses were performed in a sample of women drawn using a random number table that generated in an unpredictable, haphazard a sequence of number corresponding to the number of participants included in the study [START_REF] Annweiler | Cross-sectional association between serum vitamin D concentration and walking speed measured at usual and fast pace among older women: the EPIDOS study[END_REF]. SA (g/L) and β2m (mg/L) concentrations were determined using automated standard laboratory methods at the University Hospital of Lyon, France. The ISS was scored in 3 stages as previously described [START_REF] Ph | International staging system for Multiple Myeloma[END_REF]: stage 1 defined as β2m<3.5mg/L with SA≥3.5g/dL; stage 2 defined as β2m between 3.5-5.5mg/L with any SA level, or β2m<3.5mg/L with SA<3.5g/L; stage 3 defined as β2m≥5.5mg/L. Abnormal ISS was defined as ISS=2 or ISS=3, and ISS=1 was considered normal.

Mortality data were collected either via telephone or through a search of the French national death registry CépiDC [Centre d'épidémiologie sur les causes médicales de décès] up to October 1, 2010 [START_REF] Bailly | Mortality and profiles of community-dwelling fallers. Results from the EPIDOS cohort[END_REF]. The survival time was calculated between the date of recruitment in the EPIDOS study and the date of death or end of the follow-up.

All participants had a full examination consisting of clinical examination and structured questionnaires on socio-demographic data, health status, and physical functioning. Age, body mass index, mean arterial pressure, diabetes mellitus, hypertension, coronary heart disease, history of stroke, current use of corticosteroids, number of drugs taken per day, smoking, regular practice of physical activity, history of falls in preceding 6 months, whole-body bone mineral density, and estimated glomerular filtration rate (estimated from the Cockcroft-Gault formula: [[(140-ageyears)x weight] / creatininemol/L] x 1.04) were measured and used as potential confounders.

The study was conducted in accordance with the ethical standards set by Helsinki declaration (1983). The EPIDOS study was approved by the local ethics committee. All participants gave written informed consents.

Statistical analysis

The participants' baseline characteristics were summarized using means and standard deviations or frequencies and percentages, as appropriate. Participants were separated into 2 groups according to ISS at baseline (i.e., normal ISS versus abnormal ISS). Firstly, betweengroup comparisons were performed using the independent samples t-test or Chi-square test, as appropriate. Secondly, three consecutive Cox regression models were used to examine the associations of mortality (dependent variable) with SA, β2m and abnormal ISS adjusted on baseline characteristics (independent variables). The models produce a survival function that provides the probability of death at a given time for the characteristics supplied for the independent variables. Third, the elapsed time to death was studied by survival curves computed according to the Kaplan-Meier method and compared by the log-rank test. P-values<0.05 were considered statistically significant. All statistics were performed using SPSS (version 19.0; SPSS, Inc., Chicago, IL). Survival time after blood test correlated positively with SA concentration (r=0.16, P=0.015), and negatively with serum β2m concentration (r=-0.27, P<0.001) (Figure 1). Cox regression models revealed an inverse association between SA and mortality (fully adjusted hazard ratio (HR)=0.96, P=0.036) and a positive association between β2m and mortality (fully adjusted HR=1.30, P=0.004) (Table 2). Abnormal ISS was also associated positively with mortality (HR=2.29, P<0.001), even after adjustment for potential confounders (fully adjusted HR=3.03, P<0.001) (Table 2). Consistently, Kaplan-Meier distributions showed in Figure 2 that participants with abnormal ISS had shorter survival time than those with normal ISS (logrank P<0.001).

RESULTS

DISCUSSION

Our results show that abnormal ISS predicted, irrespective of studied potential confounders, shorter long-term survival time in community-dwelling healthy older women.

To the best of our knowledge, we provide here the first evidence of the prognostic value of the ISS in general senior population. This finding is consistent with previous studies reporting that SA and β2m are both biological prognostic factors of survival [START_REF] Goldwasser | Association of serum albumin and mortality risk[END_REF][START_REF] Corti | Serum albumin level and physical disability as predictors of mortality in older persons[END_REF][START_REF] Shinkai | Beta2-microglobulin for risk stratification of total mortality in the elderly population: comparison with cystatin C and C-reactive protein[END_REF]. Numerous epidemiological studies have showed an association between lower SA concentrations and increased morbid-mortality [START_REF] Goldwasser | Association of serum albumin and mortality risk[END_REF][START_REF] Corti | Serum albumin level and physical disability as predictors of mortality in older persons[END_REF][START_REF] Gom | Relationship between serum albumin level and aging in community-dwelling self-supported elderly population[END_REF][START_REF] Herrmann | Serum albumin level on admission as a predictor of death, length of stay, and readmission[END_REF]. The estimated increase in the odds of death ranges from 24% to 56% for each 2.5 g/L decrement in SA concentration [START_REF] Goldwasser | Association of serum albumin and mortality risk[END_REF]. This association predicts overall and cause-specific mortality including cardiovascular mortality. Several biological mechanisms have been suggested to explain the association of lower SA concentrations with increased mortality [START_REF] Goldwasser | Association of serum albumin and mortality risk[END_REF]. First, a protective effect of SA is likely based on its potential nitrovasodilator effect, which could modulate cardiovascular diseases processes.

Another protective mechanism may be related to a significant antioxidant effect of SA in blood and extracellular fluids. SA enhances the removal of reactive oxygen species involved in the pathogenesis of numerous diseases such as atherosclerosis. SA may also be directly beneficial by increasing the ability of serum to bind potential toxins. Finally, SA is an inhibitor of human endothelial apoptosis and lower SA concentrations may result in a general increased vascular permeability leading to tissue damage [START_REF] Goldwasser | Association of serum albumin and mortality risk[END_REF][START_REF] Corti | Serum albumin level and physical disability as predictors of mortality in older persons[END_REF][START_REF] Herrmann | Serum albumin level on admission as a predictor of death, length of stay, and readmission[END_REF]. Like SA, higher β2m has been associated with increased morbi-mortality while aging, independent of any history of MM [START_REF] Shinkai | Beta2-microglobulin for risk stratification of total mortality in the elderly population: comparison with cystatin C and C-reactive protein[END_REF][START_REF] Bataille | Beta-2-microglobulin in myeloma: optimal use for staging, prognosis, and treatment. A prospective study of 160 patients[END_REF][START_REF] Vincent | Serum beta2-microglobulin in hemodialyzed patients[END_REF][START_REF] Erez | Increased production of B2-microglobulin after heart transplantation[END_REF][START_REF] Annweiler | Plasma Beta-2 Microglobulin as a Marker of Frailty in Older Adults: A Pilot Study[END_REF][START_REF] Shinkai | Predictors for the onset of functional decline among initially non-disabled older people living in a community during a 6-year follow-up[END_REF]. Firstly, greater levels of serum β2m have been reported in healthy older adults compared to younger ones [START_REF] Vincent | Serum beta2-microglobulin in hemodialyzed patients[END_REF][START_REF] Shinkai | Predictors for the onset of functional decline among initially non-disabled older people living in a community during a 6-year follow-up[END_REF]. Secondly, β2m is an established biomarker of disease activity not only in malignancies and chronic renal dysfunction, but also in autoimmune affections and infections [START_REF] Bataille | Beta-2-microglobulin in myeloma: optimal use for staging, prognosis, and treatment. A prospective study of 160 patients[END_REF][START_REF] Erez | Increased production of B2-microglobulin after heart transplantation[END_REF][START_REF] Annweiler | Plasma Beta-2 Microglobulin as a Marker of Frailty in Older Adults: A Pilot Study[END_REF][START_REF] Shinkai | Predictors for the onset of functional decline among initially non-disabled older people living in a community during a 6-year follow-up[END_REF]. Thus, an increase in serum β2m concentration reflects pathological aging across multiple physiologic systems. In line with this, increased β2m concentrations were used as a marker of frailty phenotype in geriatric inpatients [START_REF] Annweiler | Plasma Beta-2 Microglobulin as a Marker of Frailty in Older Adults: A Pilot Study[END_REF]. Thirdly, a strong association has been reported among older adults between increased serum β2m concentration and higher rate of all-cause mortality, specifically cardiovascular mortality [START_REF] Shinkai | Beta2-microglobulin for risk stratification of total mortality in the elderly population: comparison with cystatin C and C-reactive protein[END_REF].

All these findings suggest that the combination of SA with β2m adequately explores the balance of inflammation proteins and constitutive proteins, and may reflect the physiological reserves available to the senior, making it an "aging system". Some limitations of our study should be considered. Firstly, the study cohort was restricted to relatively vigorous older women with interest in personal health issues who may be unrepresentative of older adults in general. Secondly, although we were able to control for important characteristics that could modify the association between ISS and mortality, residual potential confounders such as the presence of inflammatory systemic diseases or the causes of deaths might still be present. Thirdly, data were recorded before 2000. It is possible, though unproven, that the profiles and the risks of the participants have changed since then, as well as the implementation and enforcement of various prevention programs. The present results show thus trends that would better be confirmed by an independent survey in another cohort.

In conclusion, abnormal ISS predicted shorter survival time in community-dwelling healthy older women. Further studies with different cohorts are warranted to corroborate this finding, and assess the value of the ISS as an "aging system" and a universal marker of frailty phenotype in older adults.

Figure 2 .

 2 Figure 2. Kaplan-Meier estimates of the cumulative probability of participants' survival

  All 230 participants (mean age at baseline, 80.4±3.4years) died during the follow-up (mean, 17.6±0.2years; range, 16.8-18.3years). As shown in Table 1, women with abnormal ISS (n=24) had shorter survival time than those with normal ISS (4.9±4.3 versus 8.7±5.2years; P=0.001) and died earlier (mean age of death, 85.6±4.8 versus 89.1±5.6years; P=0.003).

Table 1 .

 1 Characteristics and comparison of participants separated into two groups based on the International Staging System* (n=230)

	Whole sample (n = 230)	International Staging System Normal* Abnormal* (n = 206) (n = 24)	P-value †

ACKNOWLEDGMENTS

Investigators of EPIDOS study. Coordinators: Breart, Dargent-Molina, Meunier, Schott, Hans, and Delmas. Principal investigators: Baudoin and Sebert (Amiens); Chapuy and Schott (Lyon); Favier and Marcelli (Montpellier); Hausherr, Menkes and Cormier (Paris); Grandjean and Ribot (Toulouse).

STUDY FUNDING

This work was supported by the French Ministry of Health. The sponsor had no role in the design and conduct of the study, in the collection, management, analysis, and interpretation of the data, or in the preparation, review, or approval of the manuscript.

-Obtaining funding: Schott.

-Administrative, technical, or material support: Schott.

-Supervision: Annweiler.

Data presented as mean±standard deviation where applicable. *: normal ISS defined as ISS=1; abnormal ISS defined as ISS=2 or ISS=3; †: comparisons between participants with normal ISS and participants with abnormal ISS based on independent samples t-test or Chisquare test, as appropriate; ‡: at least one hour recreational physical activity (walking, gymnastics, cycling, swimming or gardening) per week for the past month or more; ||: calculated from the Cockcroft formula; P-value significant (i.e., <0.05) indicated in bold.

CONFLICT OF INTEREST

All other authors state that they have no conflicts of interest with this paper.

CONTRIBUTIONS OF THE AUTHORS

Annweiler has access to all the study data, takes responsibility for the accuracy of the analysis, and has authority over manuscript preparation and the decision to submit the manuscript for publication.

-Study concept and design: Annweiler.

-Acquisition of data: Schott.

-Analysis and interpretation of data: Annweiler, Pouzoullic.

-Drafting of the manuscript: Annweiler, Pouzoullic.

-Critical revision of the manuscript for important intellectual content: Schott, Sánchez-Rodríguez, Bataille.