Neuroendocrine control of maternal stress responses and fetal programming by stress in pregnancy

Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 1178–1191

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Neuroendocrine control of maternal stress responses and fetal programming by stress in pregnancy Paula J. Brunton, John A. Russell ⁎ Laboratory of Neuroendocrinology, Centre for Integrative Physiology, Hugh Robson Building, George Square, University of Edinburgh, Edinburgh, EH8 9XD, UK

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Article history: Received 2 October 2010 Received in revised form 30 December 2010 Accepted 31 December 2010 Available online 6 January 2011 Keywords: Allopregnanolone Anxiety-type behaviour Endogenous opioid Hypothalamo-pituitary-adrenal axis Prenatal stress

a b s t r a c t The major changes in highly dynamic neuroendocrine systems that are essential for establishing and maintaining pregnancy are outlined from studies on rodents. These changes optimise the internal environment to provide the life support system for the placenta, embryo and fetus. These include automatic prevention of further pregnancy, blood volume expansion, increased appetite and energy storage. The brain regulates these changes, in response to steroid (estrogens, progesterone) and peptide (lactogens, relaxin) hormone signals. Activation of inhibitory endogenous opioid mechanisms in the brain in late pregnancy restrains premature secretion of oxytocin, and attenuates hypothalamo-pituitary-adrenal (HPA) responses to stress. This opioid mechanism is activated by allopregnanolone, a neuroactive progesterone metabolite. The significance of reduced HPA axis responses in shifting maternal metabolic balance, and in protecting the fetuses from adverse programming of HPA axis stress responsiveness and anxious behaviour in later life is critically discussed. Experimental studies showing sex-dependent fetal programming by maternal stress or glucocorticoid exposure in late pregnancy are reviewed. The possibility of over-writing programming in offspring through neurosteroid administration is discussed. The impact of maternal stress on placental function is considered in the context of reconciling studies that show offspring programming by stress in very early or late pregnancy produce similar phenotypes in the offspring. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Adaptations in the maternal brain during pregnancy serve to optimise pregnancy outcome. These ensure that an appropriate internal environment is provided; they prepare the brain to support the birth process and ensure the immediate expression of maternal nurturing behaviour and lactation postpartum; and perhaps protect the developing fetus(es) from events that potentially have long-term adverse programming effects on health in later life. These adaptations are driven by the actions of the hormones of pregnancy on the brain. The activities of multiple neuroendocrine systems are modified in

Abbreviations: α-MSH, α-melanocyte stimulating hormone; 3α-HSD, 3α-hydroxysteroid dehydrogenase; 11β-HSD1/2, 11β-hydroxysteroid dehydrogenase type 1 or 2; ACTH, adrenocorticotrophic hormone; CeA, central nucleus of the amygdala; CpG, cytosine-phosphate-guanine; CRH, corticotropin releasing hormone (factor); CRH-R1/2, CRH receptor type 1 or 2; EPM, elevated plus maze; GABA, γ-aminobutyric acid; GABAA, γ-aminobutyric acid type A receptor; GnRH, gonadotropin releasing hormone; GR, glucocorticoid receptor; HPA, hypothalamo-pituitary-adrenal; IL-1β, interleukin-1β; LPS, lipopolysaccharide; MR, mineralocorticoid receptor; mRNA, messenger ribonucleic acid; NPY, neuropeptide Y; NTS, nucleus tractus solitarii; POMC, pro-opioimelanocortin; PENK-A, pro-enkephalin-A; PNS, prenatal stress; pPVN, parvocellular paraventricular nucleus; PR, progesterone receptor; THDOC, tetrahydrodeoxycorticosterone; TIDA, tubero-infundibular dopaminergic; UC, urocortin. ⁎ Corresponding author. Tel.: + 44 131 650 2861; fax: +44 131 650 2872. E-mail address: [email protected] (J.A. Russell). 0278-5846/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2010.12.023

pregnancy to achieve these requirements (Brunton and Russell, 2008a, 2008b). In this review we will outline the major changes in hormone secretion and the internal environment that maintain pregnancy, and then focus on the mechanisms that suppress neuroendocrine stress responses in pregnancy. We will then review evidence that these adaptations do not fully protect the offspring from adverse programming by maternal stress of their HPA axis responsiveness and anxious behaviour. We consider the different sensitivities to programming between males and females at the gene expression, functional and behavioural levels. We explore an approach to over-writing programming in the offspring by applying understanding of how hypothalamo-pituitary adrenal (HPA) axis responses are suppressed in late pregnancy. Lastly, we consider how findings that maternal stress at very early stages of pregnancy and at late stages of pregnancy can have similar programming effects might be reconciled, despite the extreme differences in stage of development. Most of the details discussed in this review come from studies on laboratory rodents in their first pregnancy.

2. Hormone changes in early pregnancy The essential reactions in early pregnancy are the signalling of the state of pregnancy to ensure production of sufficient progesterone to

P.J. Brunton, J.A. Russell / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 1178–1191

enable implantation, and succour of the embryo via the endometrium, followed by the rapid development of the placenta (Douglas, 2010a). In humans, fertilisation of the ovum leads to the implanting embryo producing chorionic gonadotropin, which acts as the endocrine signal from pregnancy on the corpus luteum to sustain progesterone secretion. By the end of the first trimester, the placenta is itself the main source of increasing levels of progesterone. In rodents, copulation provides a neural stimulus that triggers the secretion of prolactin, which together with luteinising hormone, acts on the corpora lutea to maintain progesterone secretion. Eventually, placental lactogen substitutes for prolactin to maintain the corpora lutea (Soares et al., 1998), which remain the essential source of increasing production of progesterone throughout pregnancy. Progesterone, a steroid, acts on progesterone receptors (PR) after induction of their expression by estrogen, also a steroid (acting via estrogen receptors). Hence, there are multiple interactions of progesterone and estrogen that have peripheral effects to maintain pregnancy, and to prepare for lactation through actions with the lactogenic hormones on the mammary glands. Similarly, there are multiple actions of estrogen and progesterone within the brain, which they can readily enter as they are lipid soluble, where their actions result in the optimal activity of neuroendocrine systems for the maintenance and successful outcome of pregnancy (Brunton and Russell, 2010b). Some of the actions of progesterone are indirect, in that they are the result actions of neuroactive metabolites, in particular allopregnanolone; levels of allopregnanolone increase pari passu with those of progesterone through pregnancy (Concas et al., 1999). Such actions are not mediated via intracellular PR, but by interactions with neuronal membrane receptors, in particular GABAA receptors (Puia et al., 1990). Normal cyclical ovarian activity is suppressed from the start of pregnancy as a result of central feedback inhibition by the increased secretion of estrogens and progesterone, thus preventing the chance of competition from a further conception. This is considered to involve suppression by endogenous opioid of stimulation of GnRH neurons in the hypothalamus (Devorshak-Harvey et al., 1987). It would seem appropriate for the activity of kisspeptin neurons, recently established as essential mediators of feedback signals regulating GnRH neurons (Clarkson and Herbison, 2009), to be reduced in pregnancy, but kisspeptin mRNA expression in arcuate kisspeptin neurons is increased, and sensitivity to kisspeptin is high, so the suppression of ovulation is not so simple to explain (Roa et al., 2006). In addition to the production of chorionic gonadotropin and placental lactogen in pregnancy, another peptide hormone that is pregnancy-specific is relaxin, produced by the corpora lutea (Sherwood et al., 1980). Hormones of the prolactin family can enter the brain by selective transport across the blood-brain barrier, or more directly in the arcuate nucleus, to have important actions in the preparation for maternal behaviour and in feedback regulation of prolactin secretion, via selective trans-membrane receptors (Lee and Voogt, 1999; Mann and Bridges, 2001). Relaxin has central actions via receptors in the subfornical organ, which is important in the regulation of salt and water balance (Sunn et al., 2002). 3. Changes to the internal environment in pregnancy Partitioning of energy in the body is altered in pregnancy to ensure adequate fetal nutrition, increased maternal energy storage as fat, and to provide energy for the extra demands of maternal tissues. These changes involve increased appetite and decreased metabolism of energy stores, all orchestrated by appetite-regulating networks in the brain, especially the hypothalamus (Douglas et al., 2007; Ladyman et al., 2010). Increased appetite involves central stimulatory actions of progesterone and altered homeostatic mechanisms that involve neuropeptides produced and acting in the brain, and appetiteregulating peptides produced in the periphery. Hence, as pregnancy

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progresses, appetite and food intake continually increase. This results in part from a permissive role of resistance to leptin, which is secreted by adipose tissue, and enters the brain via the choroid plexus and the arcuate nucleus, where its normally important inhibitory actions on neuropeptide Y (NPY) neurons to reduce appetite are reduced as pregnancy progresses. The mechanisms of central leptin resistance are reviewed elsewhere (Ladyman et al., 2010), and involve also resistance of appetite control mechanisms to inhibition by αmelanocyte stimulating hormone (α-MSH) produced by arcuate nucleus neurons, which is a mediator of leptin actions (Ladyman et al., 2009). The changes in the internal environment include a progressively large increase in circulating blood volume, with reduced osmolarity, to support placental blood flow. This reflects increased water and sodium intake and increased water retention stimulated by greater vasopressin secretion, and salt retention caused by reduced secretion and action of atrial natriuretic peptide. All of these changes are primarily the result of action of relaxin on the osmoregulatory network in the lamina terminalis (Brunton et al., 2008a). 4. Birth and maternal behaviour Parturition is driven by oxytocin, secreted from the posterior pituitary by axon terminals of magnocellular neurons in the hypothalamic paraventricular and supraoptic nuclei. These neurons are intermittently strongly excited by positive neural feedback from the birth canal once parturition has started, so it is important that this process is not prematurely triggered (Russell et al., 2003). Central inhibitory mechanisms emerge in pregnancy that restrain premature activation of oxytocin secretion: these mechanisms are the same as those that attenuate neuroendocrine stress responses, discussed below (Brunton et al., 2008b). In addition to the peripheral oxytocin actions, oxytocin released into the brain is important in promoting maternal behaviour, and in maternal bonding with the newborn (Pedersen and Boccia, 2002; Shahrokh et al., 2010). It is also important in local positive autocrine feedback which co-ordinates burst-firing of oxytocin neurons so that they secrete oxytocin in discrete pulses (Rossoni et al., 2008). This centrally released oxytocin comes from axon terminals of centrally projecting oxytocin neurons, and from the dendrites of the magnocellular neurons (Bealer et al., 2010). Inhibitory mechanisms also restrain the central release of oxytocin in pregnancy (Russell et al., 2003). Oxytocin released into the brain during and immediately after birth acts on neural circuitry in the hypothalamus and limbic brain that has been prepared during pregnancy by actions of estrogen, progesterone and prolactin to organise the rapid expression of maternal behaviour post partum (Rosenblatt et al., 1988; Pedersen and Boccia, 2002). In the rat, oxytocin is also secreted under stress conditions (Ondrejcakova et al., 2010). Hence, it would be appropriate for this response to be suppressed in pregnancy, to avoid premature stimulation of the birth process. Indeed, oxytocin secretion comes under strong inhibition in late pregnancy by an endogenous opioid mechanism in the brain that acts to suppress responses to excitatory stimuli (Douglas et al., 1995; Brunton et al., 2006b). This mechanism is activated by allopregnanolone, the neurosteroid metabolite of progesterone, which also acts directly on oxytocin neurons to potentiate actions of GABA, until the end of pregnancy (Koksma et al., 2003). The allopregnanolone-opioid mechanism also restrains stress responses of the HPA axis, and is described in detail below. 5. The hypothalamo-pituitary-adrenal axis The predominant neuroendocrine response to exposure to a stressor (a stimulus or conditions that threaten homeostasis or survival, which may be emotional or physical) is secretion into the

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systemic circulation of glucocorticoid (cortisol in humans and many other species, corticosterone in rodents). 5.1. Roles of glucocorticoids in stress Glucocorticoid hormones have multiple functions, exerted via intracellular glucocorticoid receptors (GR) and higher affinity mineralocorticoid receptors (MR). GR are more widely expressed than MR, and both are expressed in the brain: GR is essentially expressed throughout the brain, and MR rather exclusively in the limbic system, especially the hippocampus (de Kloet et al., 1998; Joëls et al., 2008; de Kloet et al., 2009). Systemic actions of glucocorticoids include mobilisation of glucose from energy stores, catabolic actions on tissue protein, organisation of fat stores (Dallman et al., 1999), and modulating actions on immune system responses (Pruett, 2001). Glucocorticoids also have actions in the brain that direct appropriate cognitive and behavioural processes (Olijslagers et al., 2008; Karst et al., 2010), including regulation of appetite (la Fleur et al., 2004). These actions are all interpretable as providing essential metabolic support for physiological and behavioural responses to stress. However, if the secretion of glucocorticoid continues at a high level and is sustained or repeated, then the consequences can be damaging; such circumstances are considered to increase allostatic load (McEwen, 2008). 5.2. Hypothalamus and anterior pituitary The secretion of glucocorticoid in response to a stressor is stimulated by increased ACTH secretion by anterior pituitary gland corticotrophs, which in turn are stimulated by a variable mix of corticotropin releasing hormone (CRH) and vasopressin secreted in the median eminence from the axon terminals of neurons located in the hypothalamic parvocellular paraventricular nucleus (pPVN). Together these components comprise the HPA axis, which is essential for survival in the face of severe stress. The pPVN CRH/vasopressin neurons are driven by multiple neural pathways from rostral (limbic and prefrontal cortical regions) and caudal (brainstem relays, involving especially noradrenergic nucleus tractus solitarii [NTS] and serotonergic raphe neurons), which respectively process primarily emotional or physical stressors (Ulrich-Lai and Herman, 2009). The rostral inputs use glutamate and GABA as respective primary excitatory and inhibitory neurotransmitters, such that the activity of the pPVN CRH/vasopressin neurons is a resultant of the relative net activity of all these inputs (Herman et al., 2003; Jankord and Herman, 2008). Consequently, these inputs to the pPVN CRH/vasopressin neurons represent, after convergence in the rostral or caudal processing networks, the wide range of exteroceptive and interoceptive sensory modalities that can signal threats to homeostasis or survival. 5.2.1. Peptide modulators Many other neuromodulators are involved in tempering the effectiveness of the multiple inputs to the pPVN CRH/vasopressin neurons. These include a range of neuropeptides and circulating peptide hormones, which signal about specific homeostatic states; some of these are inhibitory and others are excitatory. In the present context, we and others have investigated altered roles or effectiveness of these peptides in pregnancy, especially peptides that are involved in regulation of appetite and metabolism, and endogenous opioids, as discussed below. 5.2.3. Glucocorticoid feedback Other important modulators of the HPA axis are steroids or their mediators. Glucocorticoids are predominant regulators of the HPA axis, with multi-stage actions (Bradbury et al., 1994; de Kloet et al., 2009). The classical view is that the glucocorticoid secreted when the HPA axis is stimulated has rapid negative feedback actions on the

anterior pituitary corticotrophs to inhibit ACTH secretion (Tian et al., 1999), and has similar negative feedback actions in the brain to reduce stimulation of the excitatory inputs to the pPVN CRH/vasopressin neurons (de Kloet et al., 2005). It has also been considered that glucocorticoids inhibit pPVN CRH/vasopressin neurons through genomic actions mediated by the GR that they express; however, studies show that chronic glucocorticoid exposure acts via this mechanism to stimulate CRH mRNA expression in these neurons (Laugero et al., 2002). 5.2.4. Membrane actions of glucocorticoids and endocannabinoids Rapid feedback actions can evidently involve rapid actions through GR or MR located at the neuronal cell membrane (Di et al., 2003; Karst et al., 2005; Olijslagers et al., 2008). Direct membrane actions of glucocorticoid on pPVN CRH neurons inhibit the electrical activity of these cells, providing a mechanism for rapid negative feedback (Di et al., 2003; Evanson et al., 2010). This action involves stimulation of synthesis of endocannabinoid by CRH neurons, which is released to retrogradely and presynaptically inhibit excitatory glutamate release (Di et al., 2003). In the hippocampus, glucocorticoids act via MR to facilitate glutamate release, (Karst et al., 2005) and this might inhibit pPVN CRH neurons via activation of their GABAergic input. 5.2.5. Neurosteroids Progesterone and corticosterone can be metabolised via 5αreductase and 3α-hydroxysteroid dehydrogenase (3α-HSD) to allopregnanolone (3α-hydroxy-5α-pregnan-20-one or 3α,5α-tetrahydroprogesterone; THP) and tetrahydrodeoxycorticosterone, ((3α,5β)-3,21-dihydroxypregnan-20-one; THDOC), respectively. Both 5α-reductase and 3α-HSD are expressed in the brain, so these steroid metabolites can be produced in the brain (Brunton et al., 2009). These reduced steroids are neuroactive, that is, they have nongenomic actions on neurons, potentiating inhibitory actions of GABA by prolonging the opening time of Cl− channels in GABAA receptors (Lambert et al., 2009), including in pPVN CRH neurons (D Belleli pers comm.). As THDOC and allopregnanolone are formed rapidly from corticosterone after its secretion is stimulated by stress (Purdy et al., 1991; Barbaccia et al., 2001), its actions on pPVN CRH/vasopressin neurons are likely to contribute to rapid feedback. 5.2.6. Sex differences: sex hormones Females generally show greater HPA axis responses to acute stressors than males (Handa et al., 1994). This sex difference has been attributed to an attenuating action in males of testosterone, and its neurosteroid metabolite, androstandiol (3β-diol; 5α-androstane3β,17β-diol) (Handa et al., 2009; Williamson et al., 2010), which is produced by the same enzymes that form allopregnanolone and THDOC. Androstandiol acts in the PVN, via estrogen receptor-β, to depress HPA axis responses to stress (Handa et al., 2009). Interestingly, repeated stress increases estrogen receptor β mRNA expression in the pPVN (Somponpun et al., 2004), which suggests greater inhibitory actions of androstandiol under these conditions. Restraining actions of testosterone on the HPA axis are mediated through androgen receptor in the medial preoptic nucleus (Williamson and Viau, 2008; Williamson et al., 2010). Conversely, in females estradiol is regarded as responsible for the greater HPA axis responses to stress than those seen in males (Weiser and Handa, 2009). Specifically, estradiol, acting in the PVN via estrogen receptor-α, interferes with glucocorticoid negative feedback inhibition (Weiser and Handa, 2009). 6. Changes in the maternal HPA axis in late pregnancy There is a progressive decrease in the amplitude of the ACTH and corticosterone circadian rhythms as pregnancy progresses in the rat (Atkinson and Waddell, 1995). Importantly, there is a striking

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reduction in the responsiveness of the HPA axis to a variety of stressors (emotional and physical), which is most marked near the end of pregnancy (Neumann et al., 1998). We have studied the mechanisms involved, taking the detailed information about how the HPA axis is regulated without pregnancy, as discussed above, as a platform.

Despite the rather quiescent state of the maternal HPA axis towards the end of pregnancy, ACTH responses to combined CRH and vasopressin administration are indistinguishable from the response in virgin rats (Ma et al., 2005), so any reduced responses to stress in pregnancy must be a result of reduced central drive to the anterior pituitary corticotrophs.

6.1. Approaches to studying HPA axis function

6.3. HPA axis stress responsiveness in pregnancy

We have used well-established methods to evaluate HPA axis function. To compare basal activity between pregnant and nonpregnant states, or to follow changes with stage of pregnancy, ACTH and glucocorticoid concentrations were measured by specific assays in small serial blood samples, taken under conditions that take account of the marked circadian rhythm of secretion and to minimise stress, unless this is a feature of the study. Secretory responses of these hormones to acute specific emotional or physical stressors were similarly measured. In our studies we used restraint stress (confinement in a plastic tube for 30 min) as an emotional stressor, intravenous injection of interleukin-1β (a pro-inflammatory cytokine, at a low dose, 500 ng/kg) as an infection mimic and as a physical stressor or social stress (explained below in the section on fetal programming) as a combined physical and emotional stressor. To evaluate activity or activation of neurons that regulate the HPA axis, we have used in situ hybridisation to measure changes in expression of mRNAs of interest, or Fos immunocytochemistry. Expression level of mRNAs for the principal peptide hormones of the HPA axis changes rapidly after stimulation by stress, so providing readouts of recent activation.

From pregnancy day 15 in the rat HPA axis responses to an acute emotional stressor (placement on the elevated plus maze; EPM) decrease as term approaches (Neumann et al., 1998), and this applies to other emotional stressors, e.g. restraint (da Costa et al., 1996; Brunton et al., 2000). Similarly, HPA axis responses to the combined emotional/physical stressor of forced swimming (Neumann et al., 1998; Ma et al., 2005) or social stress exposure (Brunton and Russell, 2010a) are also strongly attenuated. In addition, administration of peptides (orexin-A or NPY) that in virgin rats increase appetite and stimulate the HPA axis, do not stimulate the HPA axis in late pregnant rats, yet continue to stimulate food intake (Brunton and Russell, 2003; Brunton et al., 2006a, 2006b). Similarly, simulation of infection by administration of lipopolysaccharide (an endotoxin from gramnegative bacteria; LPS) or IL-1β (a pro-inflammatory cytokine produced by macrophages stimulated by LPS) strongly activates the HPA axis in virgin rats, but has little effect in late pregnant rats (Brunton et al., 2005; Fig. 1a). In all these cases, where examined, the rapid gene expression responses in the pPVN neurons (i.e. increases in CRH and vasopressin mRNA levels) to an acute stressor are markedly reduced or, in the case of IL-1β challenge, barely detectable (Brunton et al., 2005). Hence, the hyporesponsiveness of the HPA axis to emotional and physical stressors is a result of reduced central drive by the pPVN CRH/vasopressin neurons. We have found that this is not a result of lack of ability of these neurons to respond, but rather, is due to strong inhibition, as explained next.

6.2.1. CRH/vasopressin neurons in the pPVN Under basal conditions, CRH and vasopressin mRNA expression in pPVN neurons in rats are reduced late in pregnancy (Johnstone et al., 2000), and there is a modest decrease in the store of CRH in the median eminence (Ma et al., 2005). These changes indicate decreased central forward drive by CRH and vasopressin (Ma et al., 2005), and this reduced drive can explain the altered secretory pattern of ACTH and corticosterone in pregnant rats. The daily circadian increase in ACTH secretion is strongly suppressed from day 2 of pregnancy until term (Atkinson and Waddell, 1995). The anterior pituitary content of pro-opioimelanocortin (POMC) mRNA, but not the content of ACTH, is also decreased near the end of pregnancy, consistent with decreased drive by hypothalamic secretagogues (Ma et al., 2005). Decreased ACTH secretion is accompanied by a less marked, but sustained decrease in peak circulating levels of corticosterone from day 2 through to day 18 of pregnancy, after which corticosterone level increases to the pre-pregnancy level (Atkinson and Waddell, 1995). Evidently, through most of pregnancy, the fetuses and placentae are exposed to reduced circadian peak levels of corticosterone. 6.2.2. Feedback The decreased synthetic activity of the pPVN CRH/vasopressin neurons may be a late result of the reduced daily secretion of corticosterone through early to mid-pregnancy (Atkinson and Waddell, 1995), and consequent reduced chronic stimulation by glucocorticoid, mentioned above (Laugero et al., 2002). Conversely, there is some evidence for increased negative feedback, as activity of 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1, which reactivates corticosterone from its inactive metabolite, in the PVN and anterior pituitary is increased in late pregnancy, and this is expected to enhance feedback actions of glucocorticoid. However, direct testing of rapid negative feedback indicates decreased negative feedback sensitivity near the end of pregnancy (Johnstons et al., 2000). We are not aware of any studies of altered roles of endocannabinoids in HPA axis feedback in late pregnancy.

Fig. 1. Allopregnanolone (AP) induces an endogenous opioid mechanism to inhibit the stimulation of ACTH secretion by immune challenge in late pregnant rats. Blood was sampled from rats on day 21 of pregnancy and from virgins, 30 min after intravenous interleukin-1β injection (IL-1β, 0.5 mg/kg): in virgin rats this produces a strong activation of the HPA axis, reflected in a marked increase in ACTH secretion (not shown, but see Fig. 3). a) Plasma ACTH concentrations following IL-1β here are given as increases in day 21 pregnant rats as percentages of increases in control virgin rats; Left— in pregnant rats given vehicle (oil) injection 20 h and 2 h before IL-1β there was no stimulation of ACTH secretion by IL-1β, but naloxone (opioid antagonist, NLX) injection (5 mg/kg) 30 min before IL-1β restored an ACTH response; Right—inhibition of AP synthesis with finasteride (5α-reductase inhibitor, 25 mg/kg) given 20 h and 2 h before IL-1β also restored an ACTH response to IL-1β, and there was no additive effect when finasteride and NLX were both given. This indicates that in pregnancy AP induces endogenous opioid inhibition of the HPA axis. b) In virgin rats, Left—naloxone did not alter the ACTH response to IL-1β; Right—AP administration 20 h and 2 h before IL-1β reduced the ACTH response to IL-1β, and naloxone reversed this, confirming that AP activates opioid inhibition of the HPA axis. *p b 0.05 vs. other group(s). Error bars are SEM. From Brunton and Russell (2011), with permission.

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6.3.1. Mechanisms of reduced HPA responses to IL-1β in late pregnancy The rostral, limbic, networks that process emotional stressors are complex (Herman et al., 2003). By contrast, the primary pathway that mediates activation of the HPA axis by circulating IL-1β is simpler, comprising A2 noradrenergic neurons in the NTS that project directly to the pPVN (Ericsson et al., 1994) so we chose to use this model to investigate the mechanisms of suppressed HPA axis responses in late pregnancy. Peripherally injected IL-1β acts via cytokine receptors on endothelial cells in the brainstem to stimulate local prostaglandin production, which then acts on prostaglandin E2 receptors on A2 neurons to excite them, leading to noradrenaline release from A2 axons in the pPVN, which stimulates the CRH/vasopressin neurons, and hence ACTH and corticosterone secretion (Zhang and Rivest, 1999; Brunton et al., 2005). 6.3.2. Inhibition by endogenous opioid We had previously found that an endogenous opioid mechanism emerges in late pregnancy to inhibit oxytocin neurons, as revealed by administration of naloxone, an opioid receptor antagonist (Douglas et al., 1995), and we also found that naloxone restored HPA axis responses to stressors in late pregnancy, including to IL-1β (Brunton et al., 2005; Douglas et al., 1998; Fig. 1a). This finding also indicates that the pPVN CRH neurons remain capable of being stimulated in late pregnancy, as naloxone restores responsiveness to stressors within a few minutes after it is administered. We found, using Fos immunocytochemistry, that NTS neurons were still activated by IL-1β in late pregnancy, and that, by measuring noradrenaline release in the PVN with microdialysis, in late pregnant rats the release of noradrenaline stimulated by IL-1β seen in virgin rats was absent (Brunton et al., 2005). Moreover, local infusion of naloxone into the PVN restored noradrenaline release after intravenous IL-1β injection (Brunton et al., 2005). Measurement of pro-enkephalin-A (PENK-A) mRNA and μopioid receptor mRNA expression in the NTS showed increases in late pregnancy, indicating the emergence of an autoinhibitory mechanism on noradrenaline release in the PVN originating in the cell bodies of NTS neurons projecting to the PVN (Brunton et al., 2005). 6.3.3. Allopregnanolone induction of opioid inhibition The uncovering of the opioid mechanism inhibiting the HPA axis in pregnancy led us to ask what signal from the state of pregnancy activated this opioid mechanism. Having failed to replicate this with estrogen and progesterone treatment of virgin rats (Douglas et al., 2000), we tested a role for allopregnanolone, the neuroactive progesterone metabolite, which is present at high levels in the brain near the end of pregnancy (Concas et al., 1999; Brunton et al., 2009). Short-term (20 h) administration of finasteride, to block action of 5αreductase, the rate limiting enzyme in the conversion pathway from progesterone to allopregnanolone, restored HPA axis responses to IL1β in late pregnancy and when naloxone was also given it had no additive effect (Fig. 1a). This indicated that allopregnanolone induces the opioid mechanism in the NTS. Indeed, following finasteride treatment of late pregnant rats the level of pENK-A mRNA expression was decreased (Brunton et al., 2009). Furthermore, when virgin rats were given allopregnanolone, HPA axis responses to IL-1β were reduced, and this action of allopregnanolone was reversed by naloxone (Fig. 1b). Measurement of pENK-A mRNA expression in the NTS confirmed that this was increased by short-term allopregnanolone treatment in virgin rats (Brunton et al., 2009). The mechanism by which allopregnanolone increases pENK-A mRNA expression in the NTS is not clear. However, additional studies found that expression of the mRNAs for the allopregnanolone synthesising enzymes (5αreductase and 3α-hydroxysteroid dehydrogenase) in the NTS and hypothalamus is increased in late pregnancy, indicating that production and action of allopregnanolone in these locations is important (Brunton et al., 2009). How the expression of these enzymes is regulated is not clear. Clearly, any stimulatory actions of increased

estrogen levels, as seen in virgin rats (Weiser and Handa, 2009), on HPA axis stress responses in late pregnancy are outweighed by inhibitory actions of allopregnanolone. 6.4. Conclusions and implications of reduced HPA axis stress responses in late pregnancy 6.4.1. Post partum reversal of HPA modulation by neurosteroid The allopregnanolone-opioid mechanism is evidently rapidly reversible, as opioid inhibition of the HPA axis disappears after giving birth, although it is still evident during birth (Wigger et al., 1999). Whether such rapid reversal, presumably a result of the dramatic fall in allopregnanolone levels following the collapse in progesterone secretion that precedes births, contributes to post partum emotional disturbances remains to be evaluated. 6.4.2. Glucocorticoids and immune mechanisms, metabolism and behaviour Glucocorticoids act to modulate immune system activity, and stimulatory actions of IL-1β, as a pro-inflammatory cytokine, on the HPA axis reflect bi-directional neuro-immune interactions (Dantzer et al., 2008). A greatly diminished rapid HPA axis response to immune challenge in late pregnancy indicates reduced modulation of immune cell activity. However, the significance of this is not clear. As glucocorticoids secreted in response to stress rapidly mobilise energy from glycogen and fat stores, reduced glucocorticoid levels during stress in late pregnancy may be expected to reduce the drain on stored energy, favouring the pregnancy and subsequent lactation. In particular, the reduced central stimulatory actions of orexin-A and NPY on the HPA axis in pregnancy with retained stimulatory actions on appetite (Brunton and Russell, 2003; Brunton et al., 2006a) can be expected to be involved in shifting energy balance in favour of the fetuses and placentae. Rapid glucocorticoid actions in the brain include modulating actions on synaptic processes, in the hippocampus and amygdala in particular, that are considered to promote organisation of appropriate behavioural responses during stress (Olijslagers et al., 2008; Karst et al., 2010). Lower circulating levels of glucocorticoid during stress in late pregnancy may be expected to be reflected in lower levels in the brain (Droste et al., 2009). This may impact on maternal stress-coping behaviour, and perhaps underlie the reduced anxious behaviour seen in late pregnancy (Wartella et al., 2003). 6.4.3. Protection against adverse fetal programming? Many studies have shown that direct manipulation of glucocorticoid levels in pregnancy, for example by administration of exogenous glucocorticoid (corticosterone or dexamethasone, a synthetic GR agonist), to expose the fetuses and placentae to high levels, results in adverse programming of the offspring (Matthews, 2002; Seckl and Meaney, 2004; Weinstock, 2005; Shoener et al., 2006; Wilcoxon and Redei, 2007). Indicators of fetal programming in such studies included increased HPA axis responsiveness and enhanced anxious behaviour (Zagron and Weinstock, 2006) in the offspring. While exposure to stress in pregnancy has been shown in many studies to result in similar adverse programming of the offspring (Brunton, 2010), it is not clear whether this is a result of exposure to high levels of glucocorticoid. We therefore proceeded to test critically the hypothesis that the reduced HPA axis responses to stress in late pregnancy may give protection against fetal programming. For this we used a psychosocial stressor in late pregnancy, as explained below. The mechanisms of HPA axis inhibition in pregnancy we have identified provide a powerful means potentially to modulate HPA axis stress responses in other circumstances. We show in the next section on fetal programming that prenatal programming effects in adult offspring can be over-ridden, at least temporarily, by neuroactive steroids.

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7. Protection of the conceptuses from adverse programming by the external environment An understanding of the importance of the quality of the internal environment during pregnancy has led in recent years to the concept of adverse prenatal programming of the offspring, in particular of the brain, by experience of a sub-optimal environment in utero. A suboptimal intra-uterine environment may be a result of adverse or stressful external environmental circumstances that the mother experiences and are signalled to the conceptuses. Such adverse programming is reflected in impact on both physical and mental health and well-being in later life, and these effects can in some respects be perpetuated by transfer to the next generation(s). Such transfer may be direct by epigenetic mechanisms, or as a result of the impact of the adversely programmed quality of maternal behaviours shown towards the next generation of offspring (Kappeler and Meaney, 2010). Consequently, the mechanisms, biological significance, prevention and amelioration of such adverse effects are of great interest. Experience in early post-natal life (e.g. maternal care) is also of great importance in the context of programming, which may evidently be either advantageous, or disadvantageous in later life. However, this aspect is outside the scope of the present review, although studies of such post-natal experiences have pointed the way to understand programming as the result of epigenetic processes (Meaney et al., 2007; Meaney, 2010). Overall, ‘programming’ is about the interaction between the environment and the rolling out of the programme in the genome (including the inherited epigenome) during development, from very early in pregnancy until birth, and during early post-natal development. At these times interactions with the mother are predominant, which can modulate the phenotype, evidently through epigenetic effects (Meaney et al., 2007; Mueller and Bale, 2008). In addition, there are striking sex differences, in that many aspects of adverse fetal programming affect males more than females (Mueller and Bale, 2008; Brunton, 2010; Dunn et al., 2010). This resembles the longestablished de-feminisation and masculinisation of the genetically male brain in development that involves epigenetic mechanisms (McCarthy et al., 2009). Susceptibility to adult illnesses, such as cardiovascular disease, metabolic syndrome, anxiety and depression can evidently be programmed before birth, and hyperactivity of neuroendocrine stress responsiveness, especially of the HPA axis may underlie these conditions (Meaney et al., 2007). At least, this concept of programming of the brain provides tenable and testable hypotheses. A corollary is that prenatal programming is a consequence of maternal stressor exposure in pregnancy, and many experimental models are predicated on this basis. It is clear that a range of adverse experiences during fetal life can have programming effects on the offspring: hence, under- or overnutrition, or maternal exposure to severe or repeated stress, is likely to modify fetal development in such a way that metabolic regulation, stress-coping and mood regulation are adversely affected in the adult offspring (Lesage et al., 2006; Levay et al., 2008). A normally modulated increased appetite and food intake and energy balance in pregnancy is therefore important. As for averting the consequences, at least to some extent, of maternal stress in pregnancy, automatic mechanisms driven by pregnancy steroid hormones (in particular the neuroactive progesterone metabolite allopregnanolone) attenuate the maternal neuroendocrine stress responses in late pregnancy that are considered to mediate adverse fetal programming (Brunton, 2010). We now focus on considering whether the reduced neuroendocrine stress responses in late pregnancy discussed above, might provide the fetuses with some protection from adverse programming by maternal stress. We consider the role of the placenta in protecting

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the fetuses from programming. We also consider how understanding the mechanisms that reset stress responses in pregnancy may lead to understanding of the phenotype of the adversely programmed offspring brain, and to some amelioration of the compromised phenotype. 8. Prenatal stress and fetal programming The perinatal period is a time of enhanced neuroplasticity and as such the early environment can influence brain development and neuronal organisation. Much research has focused on the effects of adverse early life events on the offspring in later life. It is now widely accepted that early adverse experiences or maternal stress exposure during pregnancy adversely programmes the brain, often resulting in profound alterations in the physiology and behaviour in the offspring and increasing the susceptibility of the offspring to various diseases in adulthood. In the main these types of studies have been performed in rodents using an array of different stress paradigms. In rodents prenatal stress (PNS) exposure has been associated with low birth weight (Gotoh et al., 2005; Augustine et al., 2008; Brunton and Russell, 2010a), increased anxiety- and depressive-like behaviours (Vallee et al., 1997; Morley-Fletcher et al., 2004; Poltyrev et al., 2005; Abe et al., 2007; Hellemans et al., 2010; Brunton and Russell, 2010a), hyperactivity of the HPA axis (Takahashi and Kalin, 1991; Weinstock et al., 1992; McCormick et al., 1995; Koehl et al., 1997, 1999; Koenig et al., 2005; Fan et al., 2009; Brunton and Russell, 2010a), impaired neural development (Lemaire et al., 2000), cognition (Lemaire et al., 2000), spatial memory (Yaka et al., 2007) and social/reproductive behaviours (Holson et al., 1995; Frye and Orecki, 2002), insulin resistance (Nilsson et al., 2001), increased hypertensive responses to stress (Igosheva et al., 2004) and diet-induced obesity (Tamashiro et al., 2009). The studies above used a variety of different maternal stress paradigms to investigate the consequences for the offspring. We have recently developed an ethologically relevant rodent social stress model in our laboratory to study the effects of stress exposure during pregnancy on the stress responsivity and behaviour in the offspring (Brunton and Russell, 2010a). We propose this model more accurately reflects the types of stress pregnant women and commercial livestock are likely to encounter, which are generally social-type stressors (Bjorkqvist, 2001; Russell and Brunton, 2008). In our model, pregnant rats are exposed to an unfamiliar and therefore aggressive lactating rat (days 2–8 of lactation) for 10 min/day on five consecutive days during the last week of pregnancy (days 16–20). 8.1. Effects of prenatal stress on HPA axis regulation in the offspring Reports on the effects of prenatal stress exposure on basal activity of the HPA axis in the offspring are inconsistent and depend upon their sex and age, on the species, the prenatal stress model used (including the predictability, frequency and severity of the stress) and the stage of pregnancy when applied (Brunton, 2010). However, findings from studies investigating the effects of prenatal stress on HPA axis responses to acute stress in the offspring are more consistent and generally PNS offspring display exaggerated HPA axis responses to stress in adulthood (Peters, 1982; Fride et al., 1986; Takahashi and Kalin, 1991; Weinstock et al., 1992; McCormick et al., 1995; Bosch et al., 2007; Brunton and Russell, 2010a) and the responses are prolonged compared with controls (Henry et al., 1994) (Barbazanges et al., 1996; Morley-Fletcher et al., 2003; Brunton and Russell, 2010a), indicating impaired rapid negative feedback. 8.1.1. Sex differences Both the male and female offspring of mothers exposed to social stress during pregnancy using our PNS model display exaggerated

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ACTH and corticosterone responses to acute stress in adulthood, and this is the case for both physical (e.g. immune challenge) (Brunton and Russell, 2010a) and psychological (e.g. restraint) stressors (Brunton and Russell, 2010a). However, there are sex differences in the underlying mechanisms. The greater ACTH response to IL-1β in PNS males than in control males was reflected in a greater POMC mRNA response in the anterior pituitary 4 h after IL-1β, consistent with stronger drive by the secretagogues, CRH and vasopressin, released by the pPVN neurons

(Brunton and Russell, 2010a; Fig. 2a). This was indicated also by the greater levels of CRH mRNA and vasopressin mRNA expression 4 h after IL-1β in the PNS males than the controls (Fig. 2c, e). Notably, the expression levels of these genes showed sex differences: by contrast with the males, the level of POMC mRNA in the anterior pituitary 4 h after IL-1β was not different between female PNS and female control rats (Fig. 2b), and there was only a tendency toward a significant difference between PNS and control females in the level of vasopressin mRNA after IL-1β (Fig. 2f). Nonetheless, the

Fig. 2. Sex differences in HPA axis gene expression responses to immune challenge in offspring of prenatally stressed (PNS) mothers. From days 16 to 20 of pregnancy, rats were placed individually for 10 min each day with an aggressive lactating rat; controls (CON) remained in their own cages. HPA axis responses of the adult offspring to interleukin-1β injection (IL-1β, 0.5 mg/kg) were tested: ACTH and corticosterone responses of PNS offspring were greater than responses of CON offspring (data not shown; see Fig. 3). Measurement of levels of POMC (ACTH prohormone) mRNA in the anterior pituitary (a, b), and CRH (c, d) and arginine vasopressin (AVP; e, f) mRNA expression in the parvocellular paraventricular nucleus (pPVN) 4 h after IL-1β injection showed less stimulation, relative to CON offspring, in PNS females than in PNS males. a), b) POMC mRNA level in PNS males was increased more than in CON males; PNS and CON females showed similar levels; c), d) CRH mRNA level was greater in PNS than in CON offspring, but the difference was greater in males; e), f) AVP mRNA level was greater in PNS than in CON offspring in males, with no significant difference in females. Photomicrographs are typical in situ hybridisation autoradiographs of CON and PNS male transverse pituitary and brain sections; dotted lines indicate areas where silver grain (black) density was measured as an index of mRNA level. AP: anterior pituitary, IL: intermediate lobe, 3 V: third ventricle. Marker bars: 500 μm (pituitary), 100 μm (pPVN). *p b 0.05 vs. CON. Error bars are SEM. Adapted from Brunton and Russell (2010a), with permission.

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level of CRH mRNA expression after IL-1β was greater in PNS females than in controls (Fig. 2d). These sex differences indicate a greater programming action of prenatal stress on central HPA axis control mechanisms in males than females, which might reflect a greater deviation from the more subdued stress responses in control males compared with control females, discussed above (see 5.2.6). Nonetheless, the enhanced ACTH and corticosterone responses in both males and females are evidently the result of increased drive by the CRH neurons in the pPVN as there were greater levels of CRH mRNA in the PNS rats 4 h after administration of IL-1β in both sexes (Fig. 2c, d). 8.1.2. Feedback genes The mechanisms that underpin hyperactive HPA axis responses to stress in PNS rats are not fully understood, however impaired glucocorticoid negative feedback regulation of the HPA axis may be implicated. We found a significant reduction in mineralocorticoid receptor (MR) mRNA expression in the hippocampus of PNS male and female offspring (Brunton and Russell, 2010a) in accord with findings from others where prenatal stress exposure has been associated with a decrease in MR binding (Maccari et al., 1995; Barbazanges et al., 1996). However, whereas decreased GR binding (Weinstock et al., 1992; Szuran et al., 2000), or decreased binding to both types of receptor in the hippocampus of PNS offspring has been reported (Henry et al., 1994) using other prenatal stress paradigms, in our model GR mRNA expression was not altered (Brunton and Russell, 2010a). Similarly, GR mRNA expression in the PVN was not altered in these PNS rats (Brunton and Russell, 2010a). A lack of effect on GR mRNA expression using our social stress model may reflect differences in GR and MR ontogeny. GR mRNA is first detected in the rat fetal hippocampus on embryonic day 13.5 (before we began maternal stress exposure), whereas hippocampal MR mRNA expression is undetectable until embryonic day 16.5, with a dramatic increase in MR mRNA not occurring until the last 3 days of gestation (Diaz et al., 1998). Nevertheless, decreased hippocampal MR expression might be important in any altered feedback control of the HPA axis in our PNS model, but this has not been functionally tested. Whether epigenetic mechanisms (e.g. hyper- or hypo-methylation) influence the expression of particular genes known to regulate HPA axis activity (e.g. MR) in offspring in our model in which social stress is applied late in pregnancy is not known. In adult male mice exposed to maternal chronic variable stress at early stages of pregnancy, ACTH and corticosterone secretory responses to acute stress were greater than in controls, and in the hypothalamus methylation of the CRH promoter was decreased, consistent with increased expression (Mueller and Bale, 2008). Furthermore, decreased GR mRNA expression in the hippocampus was associated with increased methylation in an exonic region of the GR gene (Mueller and Bale, 2008). In a mouse model of early life, post-natal stress the vasopressin gene is more strongly expressed in the offspring, evidently as a result of hypo-methylation (Murgatroyd et al., 2009), The effects of the quality of maternal care received by the offspring on their HPA axis responses have been attributed to histone acetylation and methylation state of the GR gene in the hippocampus (Szyf et al., 2005). Additionally, epigenetic mechanisms acting on other genes that are primarily involved in sexual differentiation of brain function more related to reproduction (McCarthy et al., 2009; G.A. Dunn et al., 2010) may underlie the greater impact of prenatal stress on male stressrelated behaviour and HPA axis function in adulthood. 8.2. Neurosteroids and the prenatally programmed phenotype Taking the cue from our studies on pregnant rats, discussed above, we have begun investigating whether exaggerated HPA axis responses in PNS offspring might involve reduced availability or action of

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neurosteroids. Adult male and female PNS rats were given allopregnanolone injections on the same schedule as before, then HPA axis responses to IL-1β were measured (Fig. 3). Allopregnanolone treatment normalised the greatly enhanced ACTH response to IL-1β in female rats (Fig. 3), but not in males (data not shown). Whether this action involves mediation by induction of an inhibitory opioid mechanism, as in pregnancy (Brunton et al., 2009), or action on GABAA receptors on pPVN neurons, needs to be investigated. Whichever is the case, comparison of the effects of administering allopregnanolone in PNS females with the reduced HPA axis responses that result from endogenous allopregnanolone in pregnancy shows the large effect neurosteroids have on the dynamic range of HPA axis stress responses (Fig. 3). In PNS male rats, we have found that androstandiol administration normalises the HPA axis responses to IL1β. Androstandiol is formed from testosterone by the same enzymes that metabolise progesterone to allopregnanolone, and like allopregnanolone it potentiates GABA actions on GABAA receptors, as well as acting via estrogen receptor-β (Handa et al., 2009; Jin and Penning, 2001; Reddy and Jian, 2010). Accordingly, it may be that in PNS adult rats the synthesis of these neurosteroids in the brain is downregulated. 8.3. Effects of prenatal stress on anxiety-type behaviour We have shown a distinct sex difference in anxiety-related behaviour in the adult offspring of rats exposed to social stress during late pregnancy (Brunton and Russell, 2010a). Male PNS rats display increased anxiety-related behaviour on the EPM, indicated by a significant reduction in the number of open arm entries and an increased latency to make their first open arm entry compared with control males (Brunton and Russell, 2010a), consistent with studies using other prenatal stress paradigms and assessing offspring behaviour using the EPM (Poltyrev et al., 1996; Vallee et al., 1997) and open-field (Wakshlak and Weinstock, 1990; Poltyrev et al., 1996). By contrast, prenatal social stress during the last week of pregnancy

Fig. 3. Allopregnanolone availability and the dynamic range of ACTH responses to interleukin-1β (IL-1β) in adult female rats depending on life experience. Peak plasma ACTH responses 15 min after IL-1β (500 ng/kg i.v.) administration in female Sprague Dawley rats, expressed as percentage of the response in virgin controls. The ACTH response was significantly reduced in day 21 pregnant (preg) rats, and partially restored by inhibiting AP production by treatment with the 5α-reductase inhibitor, finasteride (FIN; 25 mg/kg s.c., 20 h and 2 h before IL-1β, as in Fig. 1a). Conversely, administration of AP, a 5α-reduced steroid (3 mg/kg and 1 mg/kg s.c. 20 h and 2 h before IL-1β, respectively), attenuated the ACTH response to IL-1β in virgin rats (as in Fig. 1b). The ACTH response to IL-1β in adult female offspring exposed to social stress prenatally (PNS) was ca. 3-fold greater than in controls and this was normalised by prior AP treatment (as above). The dashed line indicates the ca. 11-fold dynamic range in the ACTH response to IL-1β in female rats that is evidently governed by availability of AP. n = 5–7 rats/group. Error bars are SEM. Based on data in Brunton et al. (2009), and unpublished.

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does not appear to affect anxiety-type behaviour in our PNS females (Brunton and Russell, 2010a). However, increased anxiety behaviour has been reported in the female offspring of dams exposed to social stress for a larger part of the pregnancy (d4-18), though this was in combination with a daily restraint stress (d4-10) (Bosch et al., 2007). Other studies on rats using different prenatal stress paradigms have reported less marked anxiogenic prenatal stress effects in males compared with females (Zagron and Weinstock, 2006) or increased anxiety in both males and females (Estanislau and Morato, 2006), whereas others have reported reduced anxiety in PNS females compared with control females (Zuena et al., 2008). In studies on the offspring of mice exposed to stress early in pregnancy, adult males but not females showed abnormal, maladaptive depression-like, behavioural responses in several tests (Mueller and Bale, 2008). Sex differences in anxiety-type behaviour may be a consequence of the actions of estradiol (Walf and Frye, 2007). Indeed, anxious behaviour in the females using our prenatal stress model was correlated with estrous cycle stage; with lower levels of anxiety-related behaviour at proestrus/estrus (Brunton and Russell, 2010a), consistent with previous reports (Marcondes et al., 2001). Overall, the effect of prenatal stress exposure on anxiety-type behaviour in the offspring appears to vary depending upon the species, the stress paradigm used, the stage of gestation when the stress exposure occurs, the sex of the offspring and at what age the offspring are studied.

8.3.1. Mechanisms in altered anxiety phenotype: CRH and CRH receptors in the amygdala Central administration of CRH to rats induces anxiety-type behaviours similar to those reported in PNS rats, including suppression of locomotion in the open-field (Britton et al., 1982) and aversion to the open arms of the EPM (Dunn and Berridge, 1990). These similarities indicate that increased anxiety-related behaviour in adulthood in PNS rats may be mediated via enhanced central CRH release or action. CRH expressing neurons in the central nucleus of the amygdala (CeA) are considered to be importantly involved in mediating anxious behavioural responses (Schulkin et al., 1998). Using our PNS model we have found CRH mRNA expression in the CeA is significantly increased in both male and female offspring (Brunton and Russell, 2010a) (Fig. 4), and others have reported increased CRH content and release in the amygdala of prenatally stressed male rats (Cratty et al., 1995). Similarly, in male mice (females were not studied) offspring of mothers stressed in early pregnancy the expression of CRH mRNA in

the central nucleus of the amygdala was increased, and methylation in the CRH gene promoter CpG island was reduced (Mueller and Bale, 2008). Whether methylation of the CRH gene in the amygdala in our PNS rats is similarly reduced is not known. Glucocorticoids and stress up-regulate CRH expression in the amygdala (Makino et al., 1994; Hsu et al., 1998) and central corticosteroid receptors facilitate anxiety-type behaviours (Calvo and Volosin, 2001). GR binding is greater in the amygdala in male offspring from mothers exposed to restraint during the last week of pregnancy (McCormick et al., 1995), and we found increased GR mRNA expression in the CeA in both male and female offspring in our prenatal social stress model (Brunton and Russell, 2010a). Consequently, the increased CRH mRNA level in the central amygdala in our PNS rats may be a consequence of greater drive via GR. However, this does not explain increased anxiety-related behaviour in our model as both male and female PNS offspring showed increased CRH (Fig. 4) and GR mRNA expression in the CeA, but only the PNS males exhibited an anxious phenotype (Brunton and Russell, 2010a). We have evaluated whether differences in CRH receptor expression in the amygdala may explain the sex differences in anxiety behaviour in our PNS offspring. There are two types of CRH receptors: the CRH type 1 receptor (CRH-R1) mediates stimulation of neuroendocrine stress responses and anxious behaviours, while the type 2 receptor (CRH-R2) may be important, as a mediator of urocortin (UC) II/III actions, for dampening stress responses and anxiety (Bale et al., 2000). Indeed, we have recently found the relative expression of CRHR1 (pro-anxiogenic) mRNA to CRH-R2 (pro-anxiolytic) mRNA in the amygdaloid complex is increased in PNS males, but not females, compared with controls (Brunton et al., unpubl.), which may account for the sex differences in the anxiety phenotype. We are not aware of studies on epigenetic regulation of expression of the CRH receptors.

8.4. Conclusion The hypothesis that we aimed to test was that the established reduced maternal HPA axis stress responses in pregnancy give protection to the fetuses against adverse programming, with regard to exaggerated HPA responses and anxious behaviour in the adult offspring. This is not the case for males, but partial protection may have been afforded to females as they do not show increased anxiety, although their ACTH and corticosterone responses to stress are exaggerated as in PNS males. Nonetheless, PNS females show less marked changes than PNS males after stress in the expression of key

Fig. 4. CRH mRNA expression in the central nucleus of the amygdala (CeA) in adult prenatally stressed (PNS) male and female rats. a) Quantification of CRH mRNA expression in the CeA of male (♂) and female (♀), control and PNS rats under basal conditions (expressed as the mean number of neurones positively hybridised with the CRH mRNA probe). n = 7–19 rats per group. b) Diagrammatic representation of the amygdaloid nuclei: BLA, basolateral amygdala; BMA, basomedial amygdala; CeA, central amygdala; CoA, cortical amygdala; MeA, medial amygdala; OT, optic tract. c) Representative bright-field photographs of (i) a toluidine blue stained section at low power with the CeA and optic tract (OT) indicated; representative photomicrographs of CRH mRNA hybridisation in the CeA from a PNS male rat at (ii) low and (iv) high power; (iii) representative high power photomicrograph of CRH mRNA hybridisation in the CeA from a control male rat. Scale bars: 300 μm (i, ii) and 50 μm (iii, iv). Note the greater number of clusters of silver grains overlying cells in (iv) than in (iii). *p b 0.05 vs. PNS. Error bars are SEM. Includes data adapted from Brunton and Russell (2010a), with permission.

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genes (POMC, CRH, and vasopressin) in HPA axis regulation. In the central nucleus of the amygdala, increased expression of GR mRNA and CRH mRNA are consistent with increased anxiety in PNS males, yet PNS females do not show increased anxiety. Instead, increased ratio of expression of mRNAs for CRH-R1 to CRH-R2, in PNS males but not females, may explain the sex differences in anxious behaviour after prenatal stress. To fully test our initial hypothesis it will be necessary to see if blocking allopregnanolone production in late pregnancy, which will enhance HPA axis stress responses in pregnancy, results in a female offspring phenotype more like that in males. 9. The placenta and fetal programming The mechanisms involved in transmitting the effects of maternal stress to the fetus(es) are unclear. A role for maternal glucocorticoids is commonly invoked, though other putative factors including maternal catecholamines, and a disruption in subsequent maternal behaviour may also contribute and have been discussed elsewhere (Brunton, 2010). Nonetheless, it has become clear that adverse fetal programming may occur as a result of exposing the mother to stress during very early pregnancy, before or soon after the embryos have implanted, before the brain or neuroendocrine systems have developed and before glucocorticoid receptor is expressed in the embryo (Speirs et al., 2004; Mueller and Bale, 2008). However, adverse fetal programming is also evident in offspring whose mothers were exposed to stress for the first time late in pregnancy, shortly before birth, when the brain is clearly differentiated and expresses glucocorticoid receptor (Speirs et al., 2004; Brunton, 2010). As the still developing brain and the HPA axis are functioning in late pregnancy, but not formed at the early stages of pregnancy when stress can have programming effects on endocrine function and behaviour in the offspring, there is clearly a problem in finding common mechanisms of programming at these different stages of development. The solution may be that exposure of the mother to stress may primarily modify placental function in such a way that, regardless of whether maternal stress occurs early or late in pregnancy, the maternal stress is signalled to the fetuses at a late stage in pregnancy (Mueller and Bale, 2008; Keverne, 2010). The ‘memory’ of stress the mother experiences in early pregnancy may be registered in the placenta, through the gene expression changes that have been described (in mid-pregnancy): these include altered expression of DNA methyl transferases, and of genes important in regulating growth factor availability and nutrient supply (Mueller and Bale, 2008). These changes could be the result of earlier maternal glucocorticoid action, as the fetal side of the placenta expresses GR early in gestation (Heller et al., 1986; Speirs et al., 2004; Lemberger et al., 1994). Also, dexamethasone treatment in the first week of gestation impairs social behaviour and alters startle responses in the offspring, which may be via placental actions (Kleinhaus et al., 2010). Even in the absence of further maternal stress for the rest of the pregnancy, it is possible that compromised placental function as a result of the stress in early pregnancy will lead to adverse fetal programming, perhaps through impaired nutrient supply, or by allowing programming factors (e.g. corticosterone) to access the fetus in late pregnancy. Seeking evidence of programming in early embryos of progenitors of neural circuitry that will control neuroendocrine stress responses and associated behaviours seems to be a necessary target to address this issue. It is clear that genetic sex is an important factor in susceptibility to intra-uterine fetal programming, as discussed above in relation to HPA axis responses to stress and anxious-type behaviour. Studies in mice subjected to stress early in pregnancy have shown that the placenta of males (XY-bearing), which is predominantly of fetal origin, later shows more changes in gene expression than the female placenta;

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furthermore, some of the changes are in opposite directions (Mueller and Bale, 2008). Hence, different susceptibility of the male placenta to programming of gene expression by stress in early pregnancy may have greater impact on the subsequent development of the male fetuses. 9.1. Placental 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) Inherent protection from exposure to excessive levels of natural glucocorticoid can be provided by tissue expression of 11β-HSD2, which inactivates corticosterone by catalysing conversion to 11dehydrocorticosterone (Burton and Waddell, 1999). As pregnancy progresses, 11β-HSD2 expression in the placenta increases, peaks on day 15 (Brown et al., 1996; Waddell et al., 1998) and then disappears from the placenta on day 16 of gestation in the mouse and rat, although enzyme activity persists (Brown et al., 1996; Waddell et al., 1998). Conversely, as the expression of 11β-HSD2 mRNA in the placenta decreases, the expression of 11β-HSD1, which re-activates 11-dehydrocorticosterone to corticosterone, increases, as does local corticosterone level (Speirs et al., 2004; Mark et al., 2009). Hence there is an effective placental barrier, at least through mid-pregnancy, that restricts access of endogenous glucocorticoid to the fetus (more than 90% of corticosterone passing through the maternal circulation in the placenta is inactivated) (Benediktsson et al., 1997). The widespread expression of 11β-HSD2 in the fetus in mid-gestation, and in selected tissues including some brain regions in the last week of pregnancy should provide some further protection from direct actions of maternal glucocorticoid (Brown et al., 1996). Otherwise, in late pregnancy the attenuation of maternal HPA axis responses to stress (Brunton et al., 2005) can be envisaged as potentially providing some protection against programming by glucocorticoid for the fetuses following repeated stress, at least for females (Brunton and Russell, 2010a). Multi-drug resistance protein, P-glycoprotein, is also involved in restricting access of glucocorticoids at the placenta-fetal interface until expression reportedly decreases in late gestation (Kalabis et al., 2005). Importantly, the synthetic glucocorticoids dexamethasone and betamethasone are not metabolised by 11β-HSD2 so they can pass directly to the fetuses, and partly for this reason have been used extensively to investigate programming by glucocorticoids (Matthews et al., 2002; Dunn et al., 2010) In addition, dexamethasone reduces placental P-glycoprotein level in late pregnancy, lowering the barrier to glucocorticoid transfer (Mark et al., 2009). 9.1.1. Stress and placental 11β-HSD2 Repeated stress through most of pregnancy (day 5 to 20 in rats) can increase placental 11β-HSD2 expression, and if this response fails then hippocampal neurogenesis in the adult offspring is reduced (Lucassen et al., 2009). By contrast, repeated stress only during the last week of pregnancy has been found to strongly decrease 11β-HSD2 expression, and expression of a glucose transporter gene, in male placentae (Mairesse et al., 2007). In late pregnancy acute maternal stress increases placental 11β-HSD2 expression, but prior repeated stress prevents this response, indicating that chronic stress can permit greater passage of corticosterone to the fetus (Welberg et al., 2005). Moreover, repeated stress throughout gestation reduces maternal circulating corticosterone binding globulin (CBG) levels and results in a prolonged elevation in maternal plasma corticosterone concentrations in early-mid pregnancy (Takahashi et al., 1998), indicating increased circulating levels of free corticosterone. The mechanisms that regulate placental 11β-HSD2 expression during stress are not clear. Glucocorticoid actions do not seem important, as maternal adrenalectomy on day 13 of pregnancy, treatment with dexamethasone, or pharmacological inhibition of glucocorticoid synthesis through the last week of pregnancy, or fetectomy do not alter 11β-HSD2 expression (Burton and Waddell,

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1994; Waddell et al., 1998). Nonetheless, the effect of stress is expected to functionally weaken the placental 11β-HSD2 barrier, exposing the fetus to greater levels of glucocorticoid, especially under conditions of maternal stress. The importance of the placental 11β-HSD2 barrier is shown by the programming of the offspring HPA axis and behaviour that follows chronic pharmacological inhibition of the enzyme in rats (Welberg et al., 2000), or deletion of the gene in mice (Holmes et al., 2006). These findings support many studies that have provided evidence that exposure of the fetuses to high levels of glucocorticoid leads to a programmed phenotype in the offspring (Seckl and Meaney, 2004; Dunn et al., 2010). Stress in the first week of pregnancy in mice alters 11β-HSD2 mRNA expression in the placenta at day 12, in a sex-dependent way (Pankevich et al., 2009), but it would clearly be of interest to know about effects of repeated stress in very early pregnancy on the placental barriers that regulate glucocorticoid transfer to the fetuses in late pregnancy. 10. Protective mechanisms in early pregnancy? The mechanisms by which maternal stress alters, especially in males, the expression of functionally important genes in the placenta in early pregnancy are not yet clear. Protective neuroendocrine mechanisms in early pregnancy against scripting adverse fetal programming have not been identified. From the limited studies that have been carried out, it seems that HPA axis hormonal responses to stress in early pregnancy are not attenuated (Brunton et al., 2008b; Parker and Douglas, 2010), although the pPVN CRH mRNA , but not the vasopressin mRNA response is decreased (Douglas, 2010b). Consequently, the developing placenta, which expresses GR, is likely to be exposed to high maternal levels of glucocorticoid during stress in early pregnancy (Waddell, et al., 1998). While repeated variable stress in the first week of pregnancy in mice reduced 11β-HSD2 mRNA expression in female placentae at pregnancy day 12, expression in male placentae tended to be increased (Pankevich et al., 2009). Hence, it seems that female fetuses might be exposed to more corticosterone in later pregnancy as a result of stress in early pregnancy, though whether the down-regulation of 11β-HSD2 mRNA persists to later pregnancy, and is reflected in enzyme activity, needs to be evaluated. Female offspring generally show less marked signs of programming by prenatal stress than males, as discussed above, although there are exceptions for some systems (Weinstock et al., 1992; Weinstock, 2007). Hence the hypothesis that sex-dependent prenatal programming is mediated by fetal exposure to high levels of maternal glucocorticoid seems to be inconsistent with the decreased 11β-HSD2 mRNA expression in the placentae of females after stress in early pregnancy (Pankevich et al., 2009).

10.1. Prolactin and progesterone As pointed out initially, high levels of progesterone are essential for pregnancy maintenance, and the predominant source is the corpora lutea, which are stimulated to produce progesterone by the reflexly stimulated secretion of prolactin (Douglas, 2010b). Without pregnancy, prolactin secretion is increased by stress (in rodents; Poletini et al., 2006), but it has been shown that stress in early pregnancy in mice decreases prolactin secretion, and as a consequence progesterone secretion is also decreased (Joachim et al., 2003; Douglas, 2010b). This sequence of events seems to be primarily the result of stress increasing the activity of tubero-infundibular dopaminergic (TIDA) neurons (Douglas, 2010b), which function to inhibit prolactin secretion through dopamine receptors on anterior pituitary lactotrophs. These findings raise the possibility that decreased

progesterone secretion in early pregnancy as a result of stress might be involved in altering placental gene expression. It has been shown that reduced progesterone level near the end of pregnancy leads to increased placental 11β-HSD1 expression without changing Pglycoprotein expression, which is expected to further increase local corticosterone level (Mark et al., 2009). Perhaps actions of stressinduced decreased progesterone levels in early pregnancy (Douglas, 2010b) underlie the reported altered placental 11β-HSD2 expression a few days later (Pankevich et al., 2009). 11. Perspectives The proposition that fetal programming as a consequence of maternal exposure to stress at any stage of pregnancy has adverse consequences relates to the human perspective. There is the possibility to explain propensity in childhood following prenatal stress to be mildly cognitively impaired, and in adulthood to be predisposed to experience neuro-psychiatric disorders, and cardiovascular and metabolic disease (Bale et al., 2010). Many epidemiological retrospective and prospective studies support the hypothesis that a wide range of stressors, physical and emotional, in human pregnancy act to adversely programme the offspring phenotype. Yet, in animals in the wild there is may be an advantage in showing greater HPA axis and behavioural responses, reflecting greater vigilance, to environmental threats even if there is a long-term cost (allostatic load) that reduces longevity (Oitzl et al., 2010). From this perspective, the changes in gene expression in the placenta that follow maternal stress in pregnancy can be viewed as an adaptive signalling mechanism, providing a means for the mother to communicate her exposure to environmental adversity in pregnancy to the conceptus. Thus programming of the offspring by stress may be considered a ‘predictive adaptation’ that increases their fitness to survive in a hostile world. Acknowledgement Our research is supported by the Biotechnology and Biological Sciences Research Council. References Abe H, Hidaka N, Kawagoe C, Odagiri K, Watanabe Y, Ikeda T, et al. Prenatal psychological stress causes higher emotionality, depression-like behavior, and elevated activity in the hypothalamo-pituitary-adrenal axis. Neurosci Res 2007;59: 145–51. Atkinson HC, Waddell BJ. The hypothalamic-pituitary-adrenal axis in rat pregnancy and lactation: circadian variation and interrelationship of plasma adrenocorticotropin and corticosterone. Endocrinology 1995;136:512–20. Augustine RA, Ladyman SR, Grattan DR. From feeding one to feeding many: hormoneinduced changes in bodyweight homeostasis during pregnancy. J Physiol 2008;586:387–97. Bale TL, Contarino A, Smith GW, Chan R, Gold LH, Sawchenko PE, et al. Mice deficient for corticotropin-releasing hormone receptor-2 display anxiety-like behaviour and are hypersensitive to stress. Nat Genet 2000;24:410–4. Bale TL, Baram TZ, Brown AS, Goldstein JM, Insel TR, McCarthy MM, et al. Early life programming and neurodevelopmental disorders. Biol Psychiatry 2010;68:314–9. Barbaccia ML, Serra M, Purdy RH, Biggio G. Stress and neuroactive steroids. Int Rev Neurobiol 2001;46:243–72. Barbazanges A, Piazza PV, Le Moal M, Maccari S. Maternal glucocorticoid secretion mediates long-term effects of prenatal stress. J Neurosci 1996;16:3943–9. Bealer SL, Armstrong WE, Crowley WR. Oxytocin release in magnocellular nuclei: neurochemical mediators and functional significance during gestation. Am J Physiol Regul Integr Comp Physiol 2010;299:R452–8. Benediktsson R, Calder AA, Edwards CRW, Seckl JR. Placental 11β-hydroxysteroid dehydrogenase: a key regulator of fetal glucocorticoid exposure. Clin Endocrinol 1997;46:161–6. Bjorkqvist K. Social defeat as a stressor in humans. Physiol Behav 2001;73:435–42. Bosch OJ, Musch W, Bredewold R, Slattery DA, Neumann ID. Prenatal stress increases HPA axis activity and impairs maternal care in lactating female offspring: implications for postpartum mood disorder. Psychoneuroendocrinology 2007;32: 267–78. Bradbury MJ, Akana SF, Dallman MF. Roles of type I and II corticosteroid receptors in regulation of basal activity in the hypothalamo-pituitary-adrenal axis during the

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