Prolactin, neurogenesis, and maternal behaviors

Brain, Behavior, and Immunity 26 (2012) 201–209

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Invited Review

Prolactin, neurogenesis, and maternal behaviors C.M. Larsen ⇑, D.R. Grattan Centre for Neuroendocrinology, Department of Anatomy and Structural Biology, University of Otago, Dunedin, New Zealand

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Article history: Received 15 March 2011 Received in revised form 4 July 2011 Accepted 14 July 2011 Available online 28 July 2011 Keywords: Prolactin Neurogenesis Maternal behavior Mice Epigenetic Programming

a b s t r a c t Elevated prolactin during pregnancy increases neurogenesis in the subventricular zone of the lateral ventricle (SVZ) of the maternal brain. Evidence from our laboratory has shown that low prolactin in early pregnancy, and the consequent suppression of neurogenesis in the SVZ in the adult brain, is associated with increased postpartum anxiety and markedly impaired maternal behavior. Daughters of low prolactin mothers also display increased anxiety and a significant delay in the onset of puberty, which is associated with epigenetic changes in neuronal development (see Fig. 1). This suggests that, in rodents, low prolactin in early pregnancy exerts long-term effects that influence maternal mood postpartum, and offspring development. This mini-review aims to summarize the evidence showing that the prolactininduced increase in SVZ neurogenesis during pregnancy underlies normal postpartum maternal interactions with pups. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction Reproduction in mammals has evolved to support offspring development well beyond the end of pregnancy, in most species requiring an extended period of lactation to provide nutrients to the offspring. Maternal behavior is an essential component of lactation, ensuring the mother cares for and protects the infant, in addition to delivering nutrients. ‘Maternal behavior’ is not just one behavior, but rather the term reflects a collection of nurturing behaviors, all of which promote the welfare of the infant. Many complex behavioral and neurobiological adaptations occur in the maternal brain to help the mother to cope with the demands of pregnancy and the subsequent lactation (Grattan and Kokay, 2008), including the expression of maternal behaviors after birth, suppression of aversion responses to pups, and reduction in anxiety (for review see Brunton and Russell (2011)). In many species, the anterior pituitary hormone prolactin is an essential part of the neuronal and hormonal regulation of maternal behavior. Thus, the same hormone that is responsible for the stimulation of milk production is also critically involved in mediating the central nervous system adaptations that occur during pregnancy to support lactation. Other hormones, such as oxytocin and progesterone are also essential for these changes, but the aim of the present review is to focus on one specific action of prolactin during pregnancy, the stimulation of neurogenesis in the maternal brain. Our data in mice suggest that prolactininduced neurogenesis in the mother is critical for adaptive changes

⇑ Corresponding author. E-mail address: [email protected] (C.M. Larsen). 0889-1591/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbi.2011.07.233

in mood and behavior in the postpartum period. Manipulations of prolactin during pregnancy can have dramatic effects on anxiety and maternal behavior after birth, providing a novel model for investigation of postpartum mood disorders.

2. Prolactin, a pleiotropic neuroendocrine hormone Prolactin, a 197–199 amino acid polypeptide hormone, is mainly synthesized by lactotroph cells in the anterior pituitary, however, in some species, prolactin is also synthesized by other cells in other tissues (for review see Ben-Jonathan et al. (1996)). Prolactin not only acts in an endocrine manner, but also acts in an autocrine and a paracrine manner, on many different cell types throughout the body to have over 300 known biological functions, which range from immune system regulation (Kelley et al., 2007), islet of langerhan proliferation and insulin production (Huang et al., 2009), suppression of anxiety and the stress response (Carter and Lightman, 1987; Donner et al., 2007; Torner et al., 2001), regulation of the firing of oxytocin neurons (Kokay et al., 2006; Townsend et al., 2005), stimulating neurogenesis (Shingo et al., 2003), through to promoting complex maternal behaviors (Bridges et al., 1997). Discussing these very diverse actions are beyond the scope of this mini-review (for comprehensive reviews see: BenJonathan et al. (2008), Bole-Feysot et al. (1998) and Freeman et al. (2000)), however, many of the actions of prolactin are essential for adaptation and survival of the mother and the fetus during pregnancy and lactation (for review see Grattan and Kokay (2008)). For example, during pregnancy the stress and immune responses are changed to permit embryo implantation and ensure

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fetal survival. Both prolactin and glucocorticoids are secreted at high levels throughout pregnancy. High levels of glucocorticoids increase glucose mobilization and resistance to insulin during pregnancy. While this is a beneficial action, maximizing nutrient use, it may also, potentially deleteriously, increase apoptosis of lymphocytes (Evans-Storms and Cidlowski, 1995; Wyllie, 1980). Prolactin acts through the long form of its receptor to activate signal transducer and activator of transcription 5 (Stat5) (Freeman et al., 2000; Grattan et al., 2001; Ma et al., 2005a). Stat5 binds to glucocorticoid receptors to inhibit signaling by 75% at the glucocorticoid response element (Stocklin et al., 1996). The long form of the prolactin receptor is expressed sparsely on the gluconeogenic liver and skeletal muscle tissue (Nagano and Kelly, 1994), but immune cells co-express prolactin and glucocorticoid receptors (Touraine et al., 1994). Thus, the increase in prolactin levels during pregnancy may attenuate the potentially harmful effects of the increased glucocorticoid levels on levels of apoptosis of lymphocytes. Prolactin regulates the production of cytokines by T cells (Dimitrov et al., 2004), and also augments the estrogen-derived shift of the balance of the immune response during pregnancy so that humoral immunity (T helper type II lymphocytes) predominates over cell-mediated immunity (T helper type I lymphocytes) (Cross et al., 1989; Gaunt and Ramin, 2001; Ijaz et al., 1990; Szekeres-Bartho, 2002). This shift away from cell-mediated immunity assists in the lack of immune response to the paternally-derived fetal antigens permitting implantation and continuance of the pregnancy. Thus, the adaptations that chronically elevated levels of prolactin induce during pregnancy are essential for fetal survival. However, as T helper type I lymphocytes mediate the responsiveness to viral infections, the shift in the balance of the immune system means that pregnant women are at a higher risk of some infectious diseases (Jamieson et al., 2006), and viral activation of the maternal innate immune system can lead to neuropsychiatric conditions in the offspring (De Miranda et al., 2010). Levels of hippocampal neurogenesis have been associated with mood disorders (Duman et al., 2001; Warner-Schmidt and Duman, 2006), are reduced during mid to late pregnancy (Rolls et al., 2008) a time when lactogenic stimulation changes from surges to chronically high levels, and are directly regulated by T cells (Ziv et al., 2006). While the link between prolactin and hippocampal neurogenesis is as yet unknown, as the immune system is known to influence mood, prolactin-induced alterations in immunological function during pregnancy may have important neurological implications.

3. Control of prolactin secretion In non-pregnant mammals, the basal secretion of prolactin is low. However, prolactin secretion by anterior pituitary lactotroph cells occurs at a relatively high spontaneous rate (Everett, 1956; Kidokoro, 1975), which is unique in that all other pituitary hormones require stimulating or releasing factors to promote their release. The main control of secretion, therefore, is by inhibition from the hypothalamus, through the actions of the hypothalamic hormone, dopamine (Ben-Jonathan, 1985). Dopamine is synthesized by the tuberoinfundibular dopamine (TIDA) neurons in the arcuate nucleus, and released from nerve terminals in the median eminence. Dopamine is released into the extracellular space surrounding the portal vessels, and travels via the long portal vessels to the anterior pituitary, where it binds to dopamine D2 receptors on lactotrophs, decreasing the secretion of prolactin by suppressing spontaneous action potentials in the lactotroph (for review see Freeman et al. (2000)). Dopamine is also synthesized in the rostral arcuate tuberohypophysial dopamine neurons (THDA), and is transported to the terminals in the intermediate and posterior lobes of the pituitary. In addition, dopamine is synthesized in the

Fig. 1. Diagrammatic representation of the ideas presented in this minireview. Mating induces increased secretion of prolactin, which is essential for maintenance of pregnancy in the rodent, as well as development and maturation of the mammary gland for lactation. In addition to these peripheral actions, however, prolactin induces a range of adaptive changes in the maternal brain. One striking adaptation is a prolactin-induced increase in neurogenesis in the subventricular zone (SVZ) of the maternal brain (represented here by bromodeoxyuridine-labeled recently-dividing cells in the SVZ; (see Larsen and Grattan (2010b)). These new neurons are essential for adaptive changes in mood and behavior in the postpartum female, contributing to normal reproductive behavior. Interestingly, suckling of pups during lactation induces further prolactin secretion and a subsequent increase in neurogenesis on day 14 of lactation, perhaps establishing a positive feedback loop that maintains levels of neurogenesis to contribute to the long-lasting effects of maternal experience, where following the first pregnancy and lactation the mother is now programmed to respond more rapidly to offspring (maternal memory). Adequate maternal care is essential for normal development of the offspring (dashed arrow), and failure of these adaptive changes can exert adverse programming of the neonatal brain (see Larsen et al. (submitted for publication)), resulting in changes in mood, behavior and reproductive parameters. Photograph of a lactating female mouse with 3-day-old pups courtesy of Stephen Gammie (see Hasen and Gammie (2009, 2011) and Lonstein and Gammie (2002)).

periventricular-hypophysial dopamine neurons (PHDA), and is transported to terminals in the intermediate lobe of the pituitary. Dopamine from both THDA and PHDA neurons accesses the anterior pituitary via the short portal vessels, and this contributes to the physiological suppression of prolactin secretion (DeMaria et al., 1999). Prolactin binds to prolactin receptors in many tissues, including the brain (Bole-Feysot et al., 1998). For example, in mice, prolactin receptors are densely expressed in the rostral hypothalamus, but are also strongly expressed in the arcuate nucleus, ventromedial nucleus, bed nucleus of the stria terminalis, lateral septum, and choroid plexus (Brown et al., 2010). Prolactin acts directly on all three populations of neuroendocrine dopamine neurons, which express prolactin receptors (Kokay and Grattan, 2005; Lerant and Freeman, 1998). Dopamine synthesis and release are increased in response to acute or chronic increases in prolactin levels in blood (Moore, 1987), while hypoprolactinemia results in suppression of

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dopamine secretion (Arbogast and Voogt, 1991). Prolactin stimulates gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, in neuroendocrine dopamine neurons (Arbogast and Voogt, 1991), and also modulates the phosphorylation of TH resulting in increased dopamine synthesis (Ma et al., 2005b). Activation of these neurons by prolactin requires activation of the JAK2/STAT5b signal transduction pathway (Grattan et al., 2001; Ma et al., 2005a) and also involves a range of other signaling pathways (Ma et al., 2005b). Thus, prolactin controls its own secretion through a homeostatic, short-loop, negative feedback pathway, maintaining low levels of prolactin under most physiological conditions.

4. Prolactin secretion during pregnancy Pregnancy is characterized by high levels of prolactin in most mammalian species. This is achieved by different mechanisms in different species, but is likely to involve marked changes to the feedback inhibitory systems (Grattan et al., 2008). In rodents, stimulation of the cervix during mating causes serum prolactin surges to occur two times a day, throughout early pregnancy (Erskine, 1995). This has been predominantly studied in rats, where the surges reach a peak at approximately 0300 (just prior to lights on), and 1700 (just prior to lights off), which compares to low basal levels at 1200 and 2400. Similar changes have been seen in mice, with either one (Yang et al., 2009) or two surges (Larsen and Grattan, 2010a,b) seen, depending of the time of sampling. The increased prolactin is essential for maintaining the pregnancy, by providing luteotrophic support to the corpus luteum, thereby stimulating progesterone secretion (for review see Freeman et al. (2000)). In most mammals, e.g., rodents and humans, placental lactogens, molecules that are structurally similar to prolactin and bind to prolactin receptors, are produced by the placenta. Because the placenta is divorced from the critical neuroanatomical relationship between the hypothalamus and pituitary gland, the placental lactogens are not subjected to hypothalamic regulation by dopamine, and hence provide a source of lactogenic hormones throughout pregnancy that is not subject to the normal negative feedback regulation (Grattan et al., 2008). In rodents, placental lactogens bind to prolactin receptors on TIDA neurons, and increase the production of dopamine and thereby stop the surges of prolactin secretion at around mid-pregnancy (Lee and Voogt, 1999). Despite the prolonged secretion of placental lactogen during the second half of pregnancy, pituitary prolactin secretion is initiated again in late pregnancy. At this time, prolactin secretion is dissociated from TIDA neuronal activity (Andrews et al., 2001). Changing levels of ovarian steroids contribute to significant adaptive responses in the TIDA neurons, such that they no longer respond to prolactin with an increase in dopamine production (Grattan et al., 2008). These changes persist into lactation, meaning that prolactin secretion is elevated, and unrestrained by the normal feedback inhibition system. In humans, the placenta also produces an increasing amount of placental lactogens throughout pregnancy (for review see BenJonathan et al. (2008)). The amount of placental lactogen peaks in mid-pregnancy, and appears to be predominantly secreted into the amniotic fluid, but is also found in fetal and maternal body fluids, including cerebro-spinal fluid. Unlike rodents, in humans the pituitary produces prolactin in increasing amounts throughout pregnancy (for review see Ben-Jonathan et al. (2008)). The increasing prolactin levels are linked to increasing estrogen levels, which also rise from early pregnancy. As the increase in prolactin levels happens despite the presence of placental lactogens, it seems likely that TIDA neurons in humans become less sensitive to prolactin feedback early in pregnancy. Therefore, during a human

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pregnancy, there is a large increase in both pituitary-produced prolactin, and in placental lactogens.

5. More than milk production: Prolactin actions in the maternal brain It is well established that the elevated levels of prolactin and placental lactogens during pregnancy play a major role in the development and maturation of the mammary gland. However, prolactin receptors are also widespread in the brain (Bakowska and Morrell, 1997; Brown et al., 2010; Chiu et al., 1992; Pi and Grattan, 1998), providing a substrate for the increased prolactin levels to act through to potentially influence multiple brain functions (Grattan and Kokay, 2008). As illustrated by the role for prolactin as a feedback regulator of hypothalamic dopamine neurons, prolactin is able to enter the brain to influence neuronal activity. Systemic prolactin gains access to the cerebrospinal fluid (CSF) through a saturable, carrier-mediated process (Walsh et al., 1987), possibly involving prolactin binding sites in the choroid plexus. The choroid plexus contains fenestrated capillaries, but maintains a blood-CSF barrier by means of continuous tight junctions between the choroid plexus epithelial cells (Nilsson et al., 1992). Hence, blood-borne prolactin has ready access to the choroid plexus epithelial cells, which express high levels of both the short and long forms of the prolactin receptor (Augustine et al., 2003). The precise mechanism that translocates prolactin across this epithelial layer into the CSF, however, remains to be determined, and is a potentially important regulatory checkpoint. The arcuate nucleus/median eminence region may also have an incomplete blood–brain barrier, so hormones may have more direct access to neurons in this region (Faouzi et al., 2007). In the rat, as pregnancy progresses there is an increase in the level of prolactin receptor mRNA in the choroid plexus (Augustine et al., 2003; Sugiyama et al., 1996), suggesting that transport into the brain may be enhanced during pregnancy and lactation. Moreover, unctional responses to prolactin are markedly enhanced during lactation (Brown et al., 2011). Prolactin-induced signal and transduction factor 5 (pStat5), a reliable marker of prolactin action through the long form of the receptor, is only seen in nuclei where the receptor is detected (Brown et al., 2010, 2011). In many nuclei, particularly in the hypothalamus, there is an increase in pStat5 expression during lactation (Brown et al., 2011). The increase occurs without a concomitant increase in prolactin receptor mRNA levels. Furthermore, during lactation pStat5 is not only expressed in the same areas of the brain where expression is seen in virgin animals, but also appears in a range of additional locations. Thus, not only are prolactin levels markedly elevated at this time, it appears that neurons that express the prolactin receptor are more responsive to prolactin. The increase in lactogenic activity in regions of the brain known to be involved in reproductive behaviors reinforces the role that these areas play in maternal behavior expression. Thus, the hyperprolactinemia of pregnancy and lactation has the capability of affecting a wide range of neuronal functions (Grattan and Kokay, 2008). Animals lacking a functional prolactin receptor exhibit profound deficits in expression of maternal care (Lucas et al., 1998), demonstrating a critical role for prolactin in mediating this process. Administration of prolactin to appropriately steroidprimed nulliparous rats significantly reduces the latency to onset of maternal behavior (Bridges et al., 1985, 1990), whereas blocking prolactin secretion with bromocriptine (Bridges and Ronsheim, 1990) significantly delays the onset of maternal behavior. These stimulatory actions of prolactin on maternal behavior can be mimicked by placental lactogen (Bridges et al., 1996). Interestingly, animals with a disrupted prolactin gene exhibit some maternal

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behavior (Horseman et al., 1997), presumably mediated through activation of prolactin receptors by placental lactogen. Consistent with what is known about the neuronal circuitry regulating maternal behavior, it appears that the action of prolactin or placental lactogen to stimulate maternal behavior is exerted in the medial preoptic nucleus (Bridges et al., 1997). Prolactin is also known to act as an endogenous anxiolytic agent, able to induce dose-dependent suppression of anxiety behaviors (Donner et al., 2007) as well as the acute stress response (Carter and Lightman, 1987; Torner et al., 2001). These actions of prolactin might contribute to the well-established suppression of anxiety-like behaviors during lactation. Prolactin also affects activity of oxytocin neurons (Kokay et al., 2006), alters food intake and responses to leptin in the brain (Augustine and Grattan, 2008; Ladyman et al., 2010), and contributes to the suppression of fertility during lactation (Grattan et al., 2007; McNeilly, 1997). One of the more striking adaptive responses reported during pregnancy was the ability of prolactin to induce neurogenesis in the maternal brain. Elevated prolactin secretion seen during early pregnancy in mice causes an increase in mitogenesis in the subventricular zone of the lateral ventricle (SVZ) of the maternal brain, resulting in increased incorporation of new neurons into the olfactory bulb (Shingo et al., 2003). We have proposed that the pleiotropic actions of prolactin in the brain represent a coordinated adaptation to pregnancy and lactation (Grattan and Kokay, 2008). The physiological changes of pregnancy put enormous new strains on the mother’s body. The endocrine changes associated with pregnancy function to coordinate the multifactorial adaptive responses required for the mother to cope with these strains (Brunton and Russell, 2011). Elevated prolactin (together with the related placental lactogens) during pregnancy and lactation is well placed to provide an essential afferent signal to communicate the reproductive state to the maternal brain, driving the constellation of adaptive responses (Grattan and Kokay, 2008).

6. Pheromone modulation of endogenous prolactin secretion To investigate the functional role(s) of prolactin in the brain during pregnancy is complicated. Use of dopaminergic drugs, such as bromocriptine, to suppress prolactin secretion during pregnancy may be ineffective, due to the presence of additional ligands to the prolactin receptor (e.g., placental lactogens) that are not regulated by these drugs. Moreover, prolactin is required to maintain corpus luteum function during pregnancy in rodents and possibly other species (Douglas, 2010). We have used a novel competitive antagonist, which has proved effective in our in vitro experiments (Ma et al., 2005a,b), but because of the high molar excess required, has not been suitable for in vivo work. Others have used a different prolactin receptor antagonist to demonstrate the role for prolactin in the brain to induce maternal behavior (Bridges et al., 2001). Prolactin receptor knockout mice are infertile, and cannot be used for pregnancy studies (although they have been used to demonstrate deficits in maternal behavior in virgin females as well as failure of lactation in heterozygotes, but brain specific effects during lactation are masked by failure of milk production) (Goffin et al., 1999). Hence, we set out to modulate endogenous prolactin secretion in mice using pheromones, and evaluate effects on maternal behaviors. Pheromones are specialized chemicals that members of a species use to communicate information to other members of that species. Pheromones act through the vomeronasal system and the main olfactory pathway (Jacobsen et al., 1998; Yoon et al., 2005) to influence a range of behaviors. Pheromonal signaling in the vomeronasal system is mediated through an ion channel under the control of the trpc2 gene. Mice lacking the trpc2 gene exhibit impaired maternal behaviors to pups (Hasen and Gammie,

2011), and also a decrease in maternal aggression to novel males (Hasen and Gammie, 2009) an important protective maternal instinct (for review see Lonstein and Gammie (2002)). Importantly, pheromones have been shown to alter prolactin secretion in mice (Li et al., 1989, 1990), which may subsequently affect maternal behaviors. Virgin mice were housed in split cages with a divider down the middle, such that the virgin maternally naïve females could have pheromonal but not physical contact with a male, or another female mouse. Because virgin mice are spontaneously maternal, and we wanted to determine whether elevated prolactin would enhance maternal behavior, we initially intended to reduce maternal responses using pheromones in mice. Our hypothesis was based on previous reports that male mice pheromones lower prolactin secretion in female mice (as seen in the Bruce effect, where introduction of a foreign male mouse during early pregnancy can suppress prolactin secretion causing termination of the pregnancy, (Bruce, 1959)). We hypothesised that the females exposed to male pheromones would show impaired maternal behavior. To our surprise, females exposed to male pheromones showed significantly enhanced maternal behavior, retrieving three pups much more rapidly (350 s) than control mice (490 s) (Larsen et al., 2008). Pheromones are bound to major urinary proteins, and hence placing urine-soaked bedding in the cage exposes the female to pheromones without the confounding effects of a companion. Hence, we replicated the exposure to male pheromones, simply by placing urine soaked bedding into the cage with the female so that she was only exposed to pheromones and not to the physical presence of another mouse, and observed exactly the same changes in maternal behavior (Larsen et al., 2008). To determine whether steroid hormones were important in this effect, we repeated the experiment with ovariectomized mice. Ovariectomy had no effect on spontaneously expressed maternal behavior, or on the advanced maternal behavior that occurred after repeated exposure to pups. In contrast, the pheromonal enhancement of maternal behavior was completely dependent on the steroid hormones being present. As the behavioral result was contrary to our expectation, we next evaluated serum prolactin levels in response to the male pheromones. Rather than showing an acute suppression of prolactin, the virgin mice exposed to male pheromones exhibited a period of sustained hyperprolactinemia from 24 to 72 h of pheromonal contact (Larsen et al., 2008). This may be due to use of males of the same strain, rather than a foreign strain, for our pheromone exposure. If the pheromone-induced increase of prolactin was blocked using bromocriptine, the enhanced maternal behavior was lost, demonstrating that the pheromone-induced rise in prolactin was essential for this effect. Moreover, administration of prolactin to individually housed animals mimicked the pheromone effect, suggesting that a rise in prolactin was sufficient to enhance maternal behavior in this species. Additional studies to characterize the time course of pheromonal contact revealed that virgin females needed 14–21 days of pheromonal contact with a male for the enhanced maternal behavior to occur. We were struck by the coincidence that this was a similar duration as a rodent pregnancy, and hence, the mechanisms might be similar to those occurring during a normal pregnancy. We were looking for an effect that was induced by prolactin in early pregnancy, that did not require the presence of fetal or placental hormones, and could establish neuronal changes that would lead to enhanced maternal behavior some 2–3 weeks later. Based on the observation that prolactin-induced an increase in neurogenesis in the maternal brain during early pregnancy, which subsequently led to increased levels of olfactory bulb interneurons 2 weeks later (Shingo et al., 2003), we hypothesised that neurogenesis might be a mechanism involved in the pheromone-induced enhancement of maternal behavior. We therefore examined whether pheromone exposure

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might induce neurogenesis in adult female mice. We found a marked increase in neurogenesis, which was dependent on the steroid hormones and mediated by the period of pheromonal-induced hyperprolactinemia (Larsen et al., 2008). This provided a potential mechanism whereby prolonged exposure to pheromones might alter behavior some 2–3 weeks later.

7. Prolactin, neurogenesis and maternal behavior Altman, in 1962, and then Kaplan in 1977, showed that neurogenesis, the production of new neurons from neural stem cells, occurred in the adult mammalian brain (Altman, 1962, 1969; Kaplan and Hinds, 1977). New neurons are generated in the subventricular zone (SVZ) adjacent to the lateral ventricle and also in the dentate gyri of the hippocampus in the adult brain (Altman, 1962, 1969; Altman and Das, 1965). The neuronal precursor cells form two distinct groups, a group of mitotically active progenitor cells (Morshead and van der Kooy, 1992), and a group of stem cells that appear to be quiescent (Morshead et al., 1994). The newly generated SVZ neuronal precursor cells migrate in chains along the rostral migratory stream (Doetsch and Alvarez-Buylla, 1996; Lois and Alvarez-Buylla, 1994) to the subependymal zone of the olfactory bulb, where they unchain (Hack et al., 2002) and migrate radially through the olfactory bulb to terminally differentiate into inhibitory interneurons. The new neurons populate and function predominantly (75–99%) as c-aminobutyric acid (GABA) interneurons in the overlying granule cell layer, but also sparsely populate the outermost layer of the olfactory bulb, the glomerular cell layer (Altman, 1969; Winner et al., 2002). By 15–30 days after migration, the neuroblasts have extensive dendritic processes throughout the layers of the olfactory bulb (Petreanu and Alvarez-Buylla, 2002). The granule cell layer inhibitory interneurons synapse predominantly onto the secondary dendrites of mitral cells, the output neurons of the olfactory bulb (Desmaisons et al., 1999). Thus, newly incorporated neurons assist in synchronizing the output pattern of the mitral cells, and affect odor processing (Bath et al., 2011). Prolactin mediates a pregnancy-induced increase in neurogenesis in the subventricular zone (Shingo et al., 2003). The increase in neurogenesis during early pregnancy would result in new mature granule cells being present in the maternal olfactory bulb at approximately the time of parturition. As there is evidence that granular cell turnover facilitates olfactory learning of novel odorants (Magavi et al., 2005; Rochefort et al., 2002), this could be important for fine tuning the olfactory response to pups and thus influencing maternal behavior. The insight provided by our pheromone experiments in virgin female mice lead to the inevitable question of whether the equivalent prolactin-induced rise in neurogenesis seen on day 7 of pregnancy in mice (Shingo et al., 2003) might also be important for expression of normal maternal behaviors after birth. In a similar manner, a pheromone-induced increase in neurogenesis has been implicated in social behavior (Mak et al., 2007). To investigate this question, we housed pregnant mice in split cages such that they could have contact with male or female pheromones throughout pregnancy, and subsequently assessed maternal behaviors including anxiety and pup retrieval on day 2 postpartum. While there was no effect of exposure to male pheromones on anxiety in postpartum females, mice housed in a split-cage with a virgin female for the duration of the pregnancy were significantly more anxious (Larsen and Grattan, 2010a). To determine whether the observed effect was specifically mediated by pheromones, rather than been triggered by the presence of a companion or an enriched environment in the split cages, we examined whether exposure to urine-soaked bedding was sufficient to observe the increase in anxiety. Once again,

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female pheromone exposure throughout pregnancy increased anxiety in the postpartum period (Larsen and Grattan, 2010a). Because we were specifically interested in assessing the impact of the heightened anxiety on maternal responsiveness, we examined whether the pheromone-induced changes in postpartum anxiety would change essential postpartum behaviors, such as maternal responsiveness to pups. Maternal behavior in the home cage to their own pups did not appear to be different in the female pheromone-exposed animals. When placed in a novel cage, however, an inherently stressful situation for rodents, the anxious mothers displayed dramatically impaired maternal behavior. Female pheromone-exposed mice did not show the normal increase in neurogenesis on day 7 of pregnancy, instead having levels that were very similar to virgin female mice. Therefore, similar to the male pheromone-exposed virgin females, normal maternal responses were dependent on a prolactin-induced increase in neurogenesis during pregnancy (Larsen and Grattan, 2010a). These data suggested that elevated prolactin levels during early pregnancy in mice might be crucial for postpartum maternal behaviors. In rodents, mating induces twice-daily surges of prolactin to maintain the function of the corpus luteum and establish the pregnancy. To reduce serum prolactin levels during this time without causing termination of the pregnancy, we administered a carefully timed and low dose of the dopamine D2-receptor agonist bromocriptine, which blocks prolactin secretion from the anterior pituitary gland. Based on our preliminary work identifying the timing of the prolactin surges in mice, bromocriptine was injected once daily for 3 days starting at 4.30 pm on day 1 of pregnancy. This rapidly truncated the afternoon prolactin surge and blocked the subsequent nocturnal surge of prolactin, but allowed a brief elevation of prolactin the following afternoon (prior to the next bromocriptine injection) that was sufficient to maintain pregnancy. Low prolactin during early pregnancy prevented the normal pregnancy-induced suppression of anxiety in mice (Neumann, 2003), resulting in mothers that spent significantly less time on the open arms of the elevated plus maze (EPM) on day 2 postpartum (10%) compared to postpartum controls (70%) (Larsen and Grattan, 2010b). Indeed, the low-prolactin animals showed levels of postpartum anxiety that were more typical of that seen in non-pregnant females. Prolactin treatment in early pregnancy completely prevented the increased anxiety seen in bromocriptine-treated postpartum mice, essentially restoring the normal pregnancy-induced suppression of anxiety. Similar to the female pheromone-exposed mice low-prolactin mice displayed dramatically impaired maternal behavior when tested in a novel cage (Larsen and Grattan, 2010b). These animals investigated the pups occasionally, but did not sit with or retrieve the pups. Instead, they made a nest some distance away from the pups and none of the mice expressed full maternal behavior within the designated 60min observation period. Remarkably, these data indicate that normal elevated prolactin levels observed very early in pregnancy act to reduce anxiety approximately 20 days later in the postpartum period. The normal pregnancy-induced increase in neurogenesis on day 7 of pregnancy (approx. 40% increase) was completely abolished in the low-prolactin mice. These data were consistent with the hypothesis that changes in SVZ neurogenesis mediated the behavioral effects of low prolactin, but were correlative only, and could not rule out the possibility that another distinct action of prolactin in the brain was involved. To specifically test the role of neurogenesis during pregnancy on postpartum anxiety and maternal behavior, groups of mice were injected i.p. with the mitotic inhibitor, methylazoxymethanol (MAM) from days 4 to 7 of pregnancy. This treatment significantly reduced neurogenesis in the SVZ and also reduced neurogenesis in the dentate gyrus of the hippocampus to almost non-detectable levels, but did not affect prolactin levels

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in the mothers. As MAM treatment significantly decreased hippocampal as well as SVZ neurogenesis there may have been affects on postpartum maternal memory and learning (Shors et al., 2002; Winocur et al., 2006). However, the prolactin-induced affects on neurogenesis were confined to the SVZ suggesting that it is the decrease in neural progenitors in the SVZ that underlay the changes in anxiety postpartum. As seen in low-prolactin animals, the MAM-treated mice displayed increased anxiety, and apparently normal maternal behavior in their home cage, but when placed into an anxiogenic situation, these mice also had severely impaired maternal behavior (Larsen and Grattan, 2010b). This strongly suggested that the increase in maternal neurogenesis during early pregnancy is essential for postpartum maternal behaviors. The original study identifying prolactin-induced neurogenesis in the SVZ suggested that the short form of the prolactin receptor was expressed in the SVZ and, therefore, might directly mediate prolactin actions on neural stem cells. In a recent study characterizing prolactin-responsive neurons in the mouse brain (Brown et al., 2010), we could not detect prolactin receptor mRNA (long or short form) in the SVZ, nor could we see prolactin-induced phosphorylation of the transcription factor STAT5 (pSTAT5), a marker of prolactin-mediated signal transduction through the long form of the receptor. These observations suggest that prolactin action on neurogenesis in the SVZ may be mediated indirectly, potentially through prolactin-sensitive afferent neurons. Based on the literature, dopamine (O’Keeffe et al., 2009), serotonin (Banasr et al., 2004; Soumier et al., 2010), or GABA (Akiba et al., 2009; Platel et al., 2007, 2008) might be good candidates for mediating prolactin action on neurogenesis in the SVZ. However, further work needs to be undertaken to identify specifically how prolactin acts to stimulate neurogenesis in the SVZ. Once these targets have been identified the potential interplay between prolactin, neurotransmitters and neurogenesis might identify novel targets to prevent or treat postpartum mood disorders. We have established that the increase in SVZ neurogenesis that occurs in early pregnancy is crucial for normal levels of anxiety postpartum (Larsen and Grattan, 2010b), but the mechanism of how these new cells affect mood is unknown. SVZ neurons migrate in the rostral migratory stream, contributing to granule cell turnover and replacement in the olfactory bulb (Imayoshi et al., 2008). The new granule cells become morphologically mature as early as 2 weeks after initial cell division in the SVZ, although they show markedly different electrophysiological responses to existing granule cells (Lledo et al., 2006). Thus, an increase in mitogenesis in the SVZ during early pregnancy would be expected to result in new mature granule cells being present in the maternal olfactory bulb at approximately the time of parturition. Consistent with this, we observed that reduced mitogenesis in the SVZ during early pregnancy in low-prolactin animals was subsequently associated with reduced numbers of BrdU-immunopositive cells in the olfactory bulb on day 2 postpartum. The olfactory bulb provides important afferent inputs into the amygdala (Walsh et al., 1996), a brain region implicated in mediating anxiety, and thus plasticity in afferent inputs may be critical for appropriate postpartum mood. In addition to the olfactory bulb, the newly generated SVZ neural progenitors may also migrate to other areas of the brain, such as the amygdala or cerebral cortex (Gould et al., 1999), and thus might contribute to plastic changes in other circuits involved in postpartum anxiety. The observation that mice with low prolactin during early pregnancy exhibit postpartum anxiety and impaired maternal interactions with pups suggested that this treatment might provide a novel model for investigation of postpartum mood disorders. During the postpartum period there is an increased incidence of dysfunctional mood disorders (Stowe and Nemeroff, 1995). Pathological anxiety, the most common postpartum mood disorder

(Matthey et al., 2003; Wenzel et al., 2003), not only disadvantages the mother, but also affects the infants’ ability to attach to the mother (Manassis et al., 1994), inflicting a substantial toll on both the mother and the baby. Attempts to understand the underlying mechanisms of postpartum mood disorders have been impeded by the lack of an appropriate animal model that induces anxiety or depression while retaining the unique hormonal changes and social stresses that occur during the peripartum period. Here, we demonstrate that exposure to female pheromones during pregnancy reduces overall prolactin levels and induces a behavioral state consistent with postpartum anxiety.

8. Maternal anxiety may also program neuronal development in the pups Elevated anxiety in a mother, such as that caused by prenatal stress, not only affects the mother, but also affects the offspring, and can lead to an increase in anxiety and a change in reproductive behaviors in offspring (Bosch et al., 2007). In our study, the female offspring from anxious (low-prolactin) mothers had dramatically delayed puberty compared to those from control mothers. These offspring also displayed increased anxiety. There were no differences in body weight at equivalent ages that could account for these effects, and no effect on reproduction once puberty had occurred. Cross-fostering experiments suggested that both hormone changes during pregnancy and the consequent changes in maternal postpartum behaviors independently and synergistically influenced infant development, significantly delaying the onset of puberty (Larsen et al., submitted for publication). Hence, the effect on puberty in the daughters of anxious mothers might be caused during fetal development, and after birth. We have now observed female offspring of the anxious mothers for three successive generations. Additional groups of offspring of generation 1 daughters were mated at 8 weeks of age with a stud male to produce generation 3 daughters. No further manipulation or assessment was performed in any of these generation 1 or 2 daughters to avoid potential confounding of the epigenetic transmission of any phenotype from generation 1 to 3 daughters. Remarkably, the generation 3 daughters continue to show the delayed puberty and elevated anxiety, suggesting epigenetic transmission of these traits from the original disturbances in pregnancy and/or during lactation in the grandmothers. Genetically modified mice lacking the functional paternally expressed gene 3 (PEG3) display significantly impaired maternal behavior (Curley et al., 2004; Li et al., 1999), and also show a significant delay in puberty (Broad et al., 2009). PEG3 is associated with apoptotic neuronal cell death during late development in rodents in hypothalamic nuclei involved in the control of reproduction (Broad et al., 2009), which is necessary for expression of sexually dimorphic reproductive behaviors (Davis et al., 1996). Hence, levels of apoptosis in the fetal brain during late gestation or immediately postpartum may be associated with reproductive function. To investigate whether maternal anxiety might influence neuronal cell death during development in the offspring, we examined levels of apoptosis in the brain of female offspring from lowprolactin, anxious mothers. There was an increase in apoptosis in a range of hypothalamic nuclei on day 4 postpartum in daughters of anxious mothers (Larsen et al., submitted for publication), the time point when some neurons associated with reproductive function undergo pruning in rodents (Broad et al., 2009). Further work is necessary to determine whether the increase in neuronal cell death in the daughters is driven by prolactin-induced changes in PEG3. However, it is possible that daughters of anxious mothers may end up with a diminished population of mature reproductive neurons, and this might impact on the timing of puberty. The observed

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epigenetic ‘‘programming’’ effect of maternal postpartum anxiety in the cross-fostered daughters suggests that that despite the apparently normal retrieval of pups shown in the home cage, the maternal behavior of the anxious mothers was not completely unaffected. Postpartum rodents display high levels of motivation to retrieve pups despite the presence of noxious stimuli (Pereira et al., 2005), and hence retrieval might be relatively insensitive to anxiety postpartum. 9. Conclusion Overall, our studies provide evidence that elevated prolactin very early in pregnancy induces an increase in neurogenesis in the SVZ of the maternal brain that is critical for normal adaptive changes in mood and behavior in the postpartum period in the mother: (1) suppression of anxiety-like behavior, and (2) maintenance of normal maternal behavior in an anxiogenic situation. Further, the elevation in prolactin in the mother is also critical for normal development in the daughters: (1) normal levels of apoptotic pruning during development, (2) normal onset of puberty, and (3) euthymic levels of anxiety (see Fig. 1). Several studies have reported anxiolytic actions of prolactin (Torner et al., 2001), apparently mediated through acute actions in the hypothalamus. However, it was very surprising that decreased serum prolactin levels for a few days in early pregnancy could initiate postpartum anxiety over two weeks later. These data suggest that prolactin-induced neurogenesis in the maternal brain early in gestation plays a critical role in establishing appropriate adaptive responses in the mother that subsequently alter behavioral responses postpartum. Failure of these adaptive changes might result in postpartum mood disorders or an inability to cope with stressful situations during lactation. During the postpartum period there is increased incidence of dysfunctional mood disorders (Munk-Olsen et al., 2006), with pathological anxiety, the most common mood disorder at this time (Matthey et al., 2003). Previous studies examining the hormonal regulation of mood during the peripartum period have focused on acute changes in hormones at that time, and no clear role of hormones in the control of mood has emerged. The present data provide insight into the effect of pregnancy hormones on anxiety, identifying a critical period much earlier in gestation than previously suspected. Hence, factors influencing prolactin (and potentially other hormones) early in pregnancy, such as stress, nutrition or medication, might have an important and previously unsuspected impact on mood in the postpartum period. References Akiba, Y., Sasaki, H., Huerta, P.T., Estevez, A.G., Baker, H., Cave, J.W., 2009. Gammaaminobutyric acid-mediated regulation of the activity-dependent olfactory bulb dopaminergic phenotype. J. Neurosci. Res. 87, 2211–2221. Altman, J., 1962. Are new neurons formed in the brains of adult mammals? Science 135, 1127–1128. Altman, J., 1969. Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J. Comp. Neurol. 137, 433–457. Altman, J., Das, G.D., 1965. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J. Comp. Neurol. 124, 319–335. Andrews, Z.B., Kokay, I.C., Grattan, D.R., 2001. Dissociation of prolactin secretion from tuberoinfundibular dopamine activity in late pregnant rats. Endocrinology 142, 2719–2724. Arbogast, L.A., Voogt, J.L., 1991. Ontogeny of tyrosine hydroxylase mRNA signal levels in central dopaminergic neurons: development of a gender difference in the arcuate nuclei. Brain Res. Dev. Brain Res. 63, 151–161. Augustine, R.A., Grattan, D.R., 2008. Induction of central leptin resistance in hyperphagic pseudopregnant rats by chronic prolactin infusion. Endocrinology 149, 1049–1055. Augustine, R.A., Kokay, I.C., Andrews, Z.B., Ladyman, S.R., Grattan, D.R., 2003. Quantitation of prolactin receptor mRNA in the maternal rat brain during pregnancy and lactation. J. Mol. Endocrinol. 31, 221–232.

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