Elsevier

Hormones and Behavior

Volume 58, Issue 5, November 2010, Pages 737-746
Hormones and Behavior

Evidence for cell proliferation in the sheep brain and its down-regulation by parturition and interactions with the young

https://doi.org/10.1016/j.yhbeh.2010.07.006Get rights and content

Abstract

Production of new neurons continues throughout life in the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus and is influenced by both endocrine and social factors. In sheep parturition is associated with the establishment of a selective bond with the young based on an olfactory learning. The possibility exists that endocrine changes at parturition together with interactions with the young modulate cell proliferation in the neurogenic zones. In the present study, we first investigated the existence of cell proliferation in sheep. Newly born cells labeled by the cell proliferation marker 5-bromo-2′-deoxyuridine (BrdU) were found in the SVZ, the main olfactory bulb (MOB) and the DG and completely co-localized with Ki-67, another mitotic marker. Forty to 50% of the BrdU-labeled cells contained GFAP suggestive of the presence of neural stem cells. Secondly, parturition with or without interactions with the lamb for 2 days, down-regulated the number of BrdU-labeled cells in the 3 proliferation sites in comparison to no pregnancy. An additional control provided evidence that this effect is specific to early postpartum period: estrus with interactions with males did not affect cell proliferation. Our results provide the first characterization of neural cell proliferation in the SVZ, the DG and unexpectedly in the MOB of adult sheep. We hypothesize that the down-regulation of cell proliferation observed in the early postpartum period could facilitate the olfactory perceptual and memory demands associated with maternal behavior by favouring the survival and integration of neurons born earlier.

Research Highlights

►Evidence for adult neurogenesis in the olfactory system and the hippocampus of sheep. ►Existence of neural stem cells in the main olfactory bulb contrary to rodents. ►Down-regulation of cell proliferation at early post-partum. ►Cell proliferation is not affected by estrus and mating.

Introduction

Since adult neurogenesis has been discovered fifty years ago in the hippocampus of rat (Altman, 1962), researchers have demonstrated that the production of new neurons continues throughout life in certain regions of the brain of many mammals, including humans (Curtis et al., 2007, Ming and Song, 2005). In rodents, this production of new cells is localized mainly in two structures: the subventricular zone (SVZ) placed on the wall of the lateral ventricles and the dentate gyrus (DG) of the hippocampus. In the subgranular layer (SGZ) of the dentate gyrus, progenitor cells proliferate and give rise to new cells. This first step of neurogenesis, called cell proliferation, is followed by a second step in which some cells survive, migrate and become fully functional granular neurons which are integrated into neuronal networks (Abrous et al., 2005, Alvarez-Buylla and Garcia-Verdugo, 2002, Ming and Song, 2005). In the SVZ, neural stem cells (B cells) nested in the sides of lateral ventricles give rise to transient amplifying cells (C cells), which differentiate into neuroblasts (A cells) (Alvarez-Buylla et al., 2002, Doetsch et al., 1997, Lledo et al., 2006, Lledo et al., 2008). These neuroblasts migrate along the rostral migratory stream (RMS) to the main olfactory bulb (MOB). Once reaching the MOB, new neurons then migrate radially to the granular and glomerular cell layers, where they differentiate into local inhibitory interneurons and integrate neural networks of the olfactory system (Lledo and Saghatelyan, 2005). In non-human primates, a proliferative activity within the MOB has been discovered in addition to that reported in the DG and SVZ (Kornack and Rakic, 2001). Moreover, recently progenitor cells have been found in the MOB of humans suggesting an active neural proliferation in the MOB itself (Bedard and Parent, 2004, Curtis et al., 2007). In sheep, cell proliferation has been recently reported in the DG of ewes, although proliferation was evidenced with only one marker of cell division (Hawken et al., 2009). In this species, a better characterization of cell proliferation in the DG, the SVZ and the MOB with additional cellular markers remains a necessary step to look for neurogenesis.

Cell proliferation and survival in the adult brain are influenced by various internal and external factors (Lledo et al., 2006). For instance physiological status associated with pregnancy and parturition has been found to modulate cell proliferation in rodents. In mice, proliferation of newly born cells was increased during the first third of gestation and during lactation in the SVZ but not in the SGZ and this effect is mediated by prolactin (Shingo et al., 2003). In the rat, a significant increase in proliferation in the SVZ was reported at the very end of gestation coincident with high plasmatic levels of oestradiol and it was hypothesized that neurogenesis is stimulated by oestradiol through its action on prolactin release (Furuta and Bridges, 2005). In contrast, cell proliferation in the DG decreased in the early postpartum period in the rat (Darnaudery et al., 2007, Leuner et al., 2007, Pawluski and Galea, 2007). This decrease is reversed by adrenalectomy and low corticosterone replacement to dams (Leuner et al., 2007).

Besides the influence of endocrine changes resulting from internal regulation on cell proliferation, social factors can regulate neurogenesis (Gheusi et al., 2009). For instance, chronic social stress decreases the rate of cell proliferation in the DG of a variety of species, including rats, mice, tree shrews and primates (Czeh et al., 2002, Gould et al., 1997, Mitra et al., 2006, van der Hart et al., 2002). On the contrary, exposure of female mice to the urine of a dominant male exerts a positive effect on cell proliferation in the SVZ and the DG (Mak et al., 2007). Likewise exposure to males doubled the number of newly born cells in the DG of ewes (Hawken et al., 2009). In the context of maternal behavior, pup exposure to nulliparous rats induced an increase in the rate of proliferation in the DG (Pawluski and Galea, 2007). However, studies on endocrine and social regulation on cell proliferation are still scarce in other models than rodents. The parturient sheep can represent a useful model in this respect, given the mechanisms activating maternal behavior in this species (Levy et al., 2004, Levy and Keller, 2009, Poindron et al., 2007). Indeed, maternal behavior in the ewe appears at parturition under the influence of oestradiol and vaginocervical stimulation and depends also on olfactory cues emanating from amniotic fluids that cover the neonate (Levy et al., 2004, Levy and Keller, 2009, Poindron et al., 2007). In addition, the establishment of a selective bond with the young relies on the learning of the olfactory cues of the lamb by its mother within 2 h of contact after parturition (Poindron et al., 2007). We hypothesized that the establishment of this individual bond together with specific endocrine changes at parturition could modulate cell proliferation in the neurogenic zones.

Therefore, in the present study we investigated the presence of cell proliferation in the adult brain of sheep and whether this proliferation could be modulated by parturition and interactions with the young. In a first experiment, we characterized neural cell proliferation in the SVZ, MOB and DG of adult nulliparous ewes, using a combination of three markers, Bromo-2-deoxyuridine (BrdU), Ki-67 and the glial fibrillary acidic protein, GFAP. In addition, Doublecortin (DCX) was used to reveal proliferating neuroblasts. Second, we studied the modulation of proliferation by parturition and maternal behavior by comparing the results in non-pregnant ewes with those in parturient ewes maintained or not with their lamb. In addition, a group of estrus ewes in contact with an active male was also studied as a control for hormonal changes and social interactions. In this group, the hormonal pattern of steroids was similar to that observed of parturition, although at lower levels and mating with the male provided social and somatosensory interactions including vaginocervical stimulation.

Section snippets

Characterization of cell proliferation in the brain of adult ewes

To evaluate cell proliferation, subjects were 6 nulliparous non-pregnant ewes in anestrous (NP group), of about 1–2 years of age, from the INRA research center in Nouzilly (Indre et Loire, France). Animals were permanently housed indoors, with free access to water and were fed with dehydrated lucerne, maize, straw and a supplement of vitamins and minerals. One week before the experiment, ewes were housed in individual pens (2 × 1 m).

One day before sacrifice, ewes received an intravenous injection

Presence of stem cells and neuroblasts in the sheep brain

BrdU-positive cells were observed in the SVZ, the MOB and the DG (Fig. 2). In frontal sections of the SVZ, BrdU-positive cells were distributed either singly or in clusters between the ependymal cell layer of the lateral ventricle and the mediolateral part of the caudate nucleus (Fig. 2C). At the caudal level, BrdU labeling was predominantly found along the lateral and medial margins of the ventricle (Fig. 2C1) while at the anterior level this labeling surrounded the most part of the ventricle

Discussion

Two main results have been obtained in this study. For the first time in the sheep brain, neural cell proliferation has been characterized in the SVZ, the DG and more unexpectedly in the MOB. Secondly, we have found that parturition and interactions with the young down-regulate cell proliferation in the 3 proliferation sites studied whereas estrus and sexual interactions with active males did not affect this proliferation.

Acknowledgments

This work was financially supported by INRA PHASE division and the PTR Institute Pasteur/INRA N°319. M. Brus was a recipient of a INRA/Région Centre grant. We particularly thank Dr. P. Poindron for his help and advice on the writing of the manuscript, N. Jouaneau, F. Cornilleau, E. Archer, and C. Moussu for their technical assistance and M. Batailler, M. Migaud for their advice on the protocol for BrdU administration.

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      Adult female sheep also have many proliferating cells in their SVZ and olfactory bulbs, but the numbers of these cells are reduced in parturient females that interact with their lambs compared to estrus ewes housed with a ram or anestrous ewes housed alone (Brus et al., 2010a, 2014). Despite having fewer newborn olfactory cells, neuroblast dendrite length is longer in parturient ewes compared to virgin ewes, but not if the lamb is removed immediately after birth (Brus et al., 2010a). Because nulliparous ewes primed with progesterone and estradiol before given acute ICV oxytocin infusions show reduced cell survival in the olfactory bulb compared to CSF-infused control ewes (Levy et al., 2019), the tremendous release of oxytocin that occurs at the birth of the lamb and necessary for the onset of maternal behavior (Levy et al., 1992) is likely responsible for the downregulation of newborn cells seen in naturally parturient ewes.

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