Neuroactive steroids in pregnancy: Key regulatory and protective roles in the foetal brain

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Review

Neuroactive steroids in pregnancy: Key regulatory and protective roles in the foetal brain Jonathan J. Hirst a,∗ , Meredith A. Kelleher a , David W. Walker b , Hannah K. Palliser a a b

School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW 2308, Australia The Ritchie Centre, Monash Institute of Medical Research, Monash University, VIC 3800, Australia

a r t i c l e

i n f o

Article history: Received 12 October 2012 Received in revised form 5 April 2013 Accepted 9 April 2013 Keywords: Stressors in pregnancy Pregnancy compromise Neonatal seizures Allopregnanolone Foetus Neuroprotection Neonate Placenta

a b s t r a c t Neuroactive steroid concentrations are remarkably high in the foetal brain during late gestation. These concentrations are maintained by placental progesterone synthesis and the interaction of enzymes in the placenta and foetal brain. 5␣-Pregnane-3␣-ol-20-one (allopregnanolone) is a key neuroactive steroid during foetal life, although other 3␣-hydroxy-pregnanes may make an additional contribution to neuroactive steroid action. Allopregnanolone modulates GABAergic inhibition to maintain a suppressive action on the foetal brain during late gestation. This action suppresses foetal behaviour and maintains the appropriate balance of foetal sleep-like behaviours, which in turn are important to normal neurodevelopment. Neuroactive steroid-induced suppression of excitability has a key role in protecting the foetal brain from acute hypoxia/ischaemia insults. Hypoxia-induced brain injury is markedly increased if neuroactive steroid levels are suppressed and there is increased seizure activity. There is also a rapid increase in allopregnanolone synthesis and hence levels in response to acute stress that acts as an endogenous protective mechanism. Allopregnanolone has a trophic role in regulating development, maintaining normal levels of apoptosis and increasing myelination during late gestation in the brain. In contrast, chronic foetal stressors, including intrauterine growth restriction, do not increase neuroactive steroid levels in the brain and exposure to repeated synthetic corticosteroids reduce neuroactive steroid levels. The reduced availability of neuroactive steroids may contribute to the adverse effects of chronic stressors on the foetal and newborn brain. Preterm birth also deprives the foetus of neuroactive steroid mediated protection and may increase vulnerability to brain injury and suboptimal development. These finding suggest replacement therapies should be explored. This article is part of a Special Issue entitled ‘Pregnancy and steroids’. © 2013 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of neurosteroid concentrations in the foetal and neonatal brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pivotal role of 5␣-reductases in the placenta and foetal brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuroactive steroids and GABAergic pathways in pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurosteroid regulation of foetal behavioural state and effects on development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of neuroactive steroids on brain growth and myelination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Allopregnanolone and the neurotrophin release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protective role of neuroactive steroids following acute injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuroprotection and suppression abnormal EEG activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurosteroid disruption following growth restricted pregnancies and preterm birth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of glucocorticoids and stress on neuroactive steroids synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuroactive steroid responses to stress in the neonatal period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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∗ Corresponding author. Tel.: +61 2 4042 0360; fax: +61 2 4921 7903. E-mail address: [email protected] (J.J. Hirst). 0960-0760/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jsbmb.2013.04.002

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1. Introduction In species that have long gestations such as the human, sheep and guinea pigs the placenta synthesises considerable amounts of progesterone over the last half of gestation [1]. Concentrations are markedly higher compared to non-pregnant levels and generally rise with advancing gestation. The production of progesterone by the placenta, and potentially the corpus lutuem in some species, has a key role in providing precursors for the synthesis of neuroactive steroids. This precursor production makes a major contribution to the synthesis of allopregnanolone (5␣-pregnane-3␣-ol-20-one) in the foetal periphery and brain, and leads to relatively high levels of this neuroactive steroid in the foetal brain during pregnancy compared to after birth [2]. Examination of the mechanisms controlling neuroactive steroid production in the foetus highlights the key role of interactions between the placenta and foetal brain in long gestation species (Fig. 1). Neuroactive steroids have a major influence over CNS activity and are essential for growth and neuronal and glial cell survival [3,4]. Progesterone has potent repair-promoting actions following traumatic brain injury in adults [5,6] and most of this action results from its metabolism to 5␣-reduced metabolites including allopregnanolone [7]. Placental precursor production and the high levels reached in the foetal brain suggest allopregnanolone is the most important protective steroid [2]. Allopregnanolone has an essential role in the suppression of excitability and reducing potentially damaging seizures [8]. Thus reduction in synthesis of allopregnanolone due to compromises during pregnancy may increase the vulnerability of foetal brain to seizure-induced damage. Studies in adult animals indicate that neuroactive steroids have an important trophic role in the brain and may contribute to repair processes after brain injury enhancing myelination and reducing apoptotic processes [9,10]. Our recent studies suggest this is also the case in the foetus. We found the suppression of allopregnanolone synthesis caused increased cell death in the brain and resulted in delayed myelination of the white matter tracts [11,12]. Changes in glial cell activation have also been identified and this may suggest changes to the maturation of oligodendrocytes [4]. These findings further support the importance of allopregnanolone to the developing brain and suggest that exposure to normal neuroactive steroid levels is critical. The loss of such support after premature birth may markedly contribute to neuromorbidity even if there is only a moderate degree of prematurity. Stressful events during pregnancy, especially those leading to transient hypoxia/ischaemia stimulate the production of allopregnanolone in the brain providing further protection and trophic support [13]. Much of this protection is lost after birth when

Fig. 1. Pathways contributing to allopregnanolone levels in foetal brain. The placenta may contribute progesterone, precursor metabolites and/or allopregnanolone directly to the brain. Both 5␣-reductases-1 and -2 are expressed in the placenta and foetal brain, although 5␣-reductase-2 may make the major contribution to activity in the foetal brain. 5␣-R1: 5␣-reductase-1; 5␣-R2: 5␣-reductase-2, and 5␣-DHP: 5␣-dihydroprogesteron.

allopregnanolone levels fall, however, the term newborn can produce neuroactive steroid responses that are neuroprotective [14]. Thus, rising allopregnanolone levels after lipopolysaccharide treatment show that the term newborn is able to increase neuroactive steroids in the brain in response to inflammatory stress, but not to the extent seen during foetal life. Premature birth and other pregnancy compromises lead to marked changes in allopregnanolone levels in the brain and therefore the exposure to adverse conditions without the benefit of neuroactive steroid-mediated protection [8]. Complications of pregnancy, including placental insufficiency, foetal growth restriction and chronic foetal hypoxaemia, are all associated with a higher risk for adverse outcomes including brain injury. These conditions may result in marked morbidity including cerebral palsies or less severe damage that may become apparent with advancing age [15]. It is now well-accepted that many of the behavioural problems of childhood and older ages arise during pregnancy [16,17]. Reduced neuroactive steroid levels in the foetal brain may contribute to these adverse outcomes [11,12]. The rise in cortisol levels that is associated with stress in pregnancy has been implicated in adverse outcome [18]. We have reported that synthetic corticosteroid administration to the foetus, supresses neuroactive steroid synthetic enzyme expression, and a similar suppression may occur following stress-induced increases in cortisol levels in the foetus [19]. The interaction between neuroactive steroid pathways and glucocorticoid levels may have a major impact on the developing brain. Furthermore some glucocorticoids and their metabolites may be neuroactive [20], however, there is little information on the potential roles of these metabolites in stress-induced pathologies or on interactions between the placenta and foetal brain.

2. Regulation of neurosteroid concentrations in the foetal and neonatal brain Progesterone and allopregnanolone are the most important neuroactive steroids present during pregnancy as they are found in remarkably high concentrations in the foetal circulation and brain, respectively [2]. Allopregnanolone levels in the foetal brain are regulated by intimate interactions between the placenta and brain itself and in the foetal sheep reach up to 400 pmol/g in some regions [2]. These levels far exceed those seen after birth 20–40 pmol/g or in adults and are higher than concentrations in the foetal sheep circulation during late pregnancy (80–100 nmol/L). Allopregnanolone levels are also elevated in human maternal [21] and foetal circulations [22], however concentrations reached in the human foetal brain are not available. The placenta produces substantial amounts of pregnenolone and progesterone during pregnancy with production far exceeding cyclic production in non-pregnancy [23]. This steroid production results in the entry of large amounts of progesterone into the maternal circulation where it not only maintains uterine quiescence and influences maternal immune function, but also influences maternal CNS activity with improvements in seizure threshold and reduced anxiety [24]. This is consistent with the metabolism of progesterone to neuroactive steroids and elevated levels of GABAA receptor agonist steroids some of which readily cross the placenta [25]. The effects on excitability are lost at birth with the removal of the placenta and decline in neuroactive steroid levels in the plasma [21,26]. Placental steroidogenesis markedly influences foetal progesterone levels, however in the sheep, the placenta metabolises much to the progesterone produced and consequently concentrations in the foetal circulation are less than those in the maternal circulation [27]. Alternative in human pregnancy more progesterone reaches foetus un-metabolised and levels in the foetal umbilical vein are considerably higher than those in the material circulation [28–31]. Both the human and sheep

Please cite this article in press as: J.J. Hirst, et al., Neuroactive steroids in pregnancy: Key regulatory and protective roles in the foetal brain, J. Steroid Biochem. Mol. Biol. (2013), http://dx.doi.org/10.1016/j.jsbmb.2013.04.002

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decrease as the supply of pregnenolone and progesterone is withdrawn after birth, indicating the importance of the placenta in maintaining neuroactive steroid levels in the foetal brain [2].

placenta produce a considerable number of metabolites that are able to serve as precursor for neuroactive steroid synthesis. The concentrations several of these metabolites are also higher in the foetal circulation compared to maternal levels [30,31]. The present of high precursors levels in the foetal circulation likely contributes, along with de novo synthesis within the brain, to the remarkably high levels of allopregnanolone in the late gestation foetal brain [32]. Metabolising enzymes in the placenta likely contribute to the levels of allopregnanolone in the plasma and brain directly although the relative contribution of enzymes within the brain, plasma and foetal periphery remains to be quantitated (Fig. 1). The enzymes required for the synthesis of progesterone and its conversion to allopregnanolone are expressed in the adult brain [33] and are also found in abundance in foetal sheep and foetal guinea pig brains from at least the second half of gestation [11,34]. P450 cholesterol side chain cleavage enzyme (P450scc) catalyses the formation of pregnenolone from cholesterol and is expressed in neurons and glial cells [35]. The metabolism of progesterone to allopregnanolone requires 5␣- and 3␣-reduction by the enzymes 5␣-reductase and 3␣-hydroxysteroid oxidoreductase (3␣HSR), respectively; 5␣-reductase enzymes irreversibly convert progesterone to 5␣-dihydroprogesterone, and these enzymes may be the major regulatory step in the synthesis of allopregnanolone both in the placenta and foetal brain [36]. P450scc and 5␣-reductase expression increase in late gestation, so that the foetal brain contains the greatest capacity of allopregnanolone synthesis at the time of birth [2,11]. Therefore, during foetal life, concentrations of these steroids in the brain are markedly augmented by production of precursors and/or allopregnanolone in the placenta. Thus, despite the sustained expression of P450scc and 5␣reductases in the neonatal brain, allopregnanolone concentrations

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3. Pivotal role of 5␣-reductases in the placenta and foetal brain There are two isoforms of 5␣-reductase, type-1 and -2. The 5␣reductase-2 isoform is expressed strongly in the foetal and neonatal rat brain [37], and we have found that both isoforms are expressed in foetal sheep and guinea pig brains [2,19]. Increased expression of 5␣-reductases occurs in parallel with rising allopregnanolone concentrations in the brain, supporting the proposition that these enzymes regulate foetal allopregnanolone synthesis [2]. The human placenta expresses both 5␣-reductase isoforms and is therefore a potential source of 5␣-reduced steroids for the foetal brain. Moreover, expression of both isoforms increases with advancing gestational age which would lead to increasing availability of 5␣dihydroprogesterone for allopregnanolone synthesis (Fig. 2 [36]). In sheep, the rapid metabolism of progesterone in the placenta itself and on entering foetal circulation leads to the release of a considerable number of metabolites [38]. These include large amounts of the 5␣- and 5␤-reduced pregnanes, including 5␤-pregnanolone and 5␣- and 5␤-pregnanediols. Some of these steroids may have marked neuroactive action and augment the action of allopregnanolone whereas others may be inactive. This further suggests that pregnanolone and pregnanediols may also be produced in the foetal brain from these metabolites. However, the role of these 5␤-pregnanes in the foetus requires further investigation to delineate actions and potential changes in profiles over gestation as well as with placental insufficiencies. In addition to progesterone,

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Gestational age (weeks) Fig. 2. Expression of 5␣-reductase-1 (upper panels) and 5␣-reductase-2 (lower panels) in human placenta with increasing gestational age. Left-hand panels show individual expression levels and right-hand panels show mean ± SEM expression in placentae obtained after preterm birth (22–36 weeks of gestational age) and term birth (37 completed weeks to 42 weeks). Values are arbitrary units relative to the expression of ␤-actin. *P < 0.05 preterm (n = 35) vs term (n = 54) expression [36].

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the sheep placenta releases considerable amounts of pregnenolone into the foetal circulation, most which is sulphated in the foetal liver [39]. In contrast, in the human large amounts of pregnenolone are produced and sulfated by the foetal zone of the human foetal adrenal glands. This results in considerable (low micromolar) concentrations of pregnenolone sulfate in the human foetal circulation [40,41]. Free pregnenolone may act as a precursor and contribute to progesterone and allopregnanolone production in the foetal brain however further study of the role of pregnenolone sulphate in neuroactive steroid production is needed.

4. Neuroactive steroids and GABAergic pathways in pregnancy Allopregnanolone and other 3␣-hydroxy pregnanes interact with the steroid-binding site on the GABAA receptor [42,43] resulting in hyperpolarisation of the post-synaptic neuron, with the overall effect on the brain being reduced excitation. These interactions have major regulatory roles in behaviour during late gestation by markedly suppressing CNS activity. Examination of the modulation of GABAA receptors by neuroactive steroids indicates marked differences in sensitivity depending on subunit arrangement. The GABAA receptor is comprised of 5 subunits which combine to form a central pore of the ion channel [44]. These subunits, and their isoforms, ␣(1–6), ␤(1–3), ␥(1–3), ␦, ␧, ␲, ␪, ␳ (1–3) lead to basis for a significant degree of heterogeneity of the GABAA receptor, with 19 subunits identified to date, and which determine sensitivity to agonists [45,46]. Composition therefore also determines neuroactive steroid binding affinity and the sensitivity of the receptor to modulation. Recent studies suggest that neuroactive steroids bind to two sites both of which are located on the ␣1 subunit with binding to one site leading to receptor activation and the other to potentiation [46,47]. Although ␣1 subunit binding is essential for steroid modulation, the presents of other subunits cause conformational changes that regulate sensitivity. The activation of the receptor requires higher steroid concentrations compared to potentiation, however, binding to both site may be required to maximally stimulate of the receptor [46,47]. While the presence of an ␣1 subunit is necessary for neuroactive steroid action at the receptors, extensive studies suggest that the presence of a ␦ subunits leads to high sensitivity to modulation by neuroactive steroids [48,49]. The subunit composition of the GABAA receptor also differs with localisation [50]. In the adult, specific regional patterns of subunit expression are seen, such as the ␣1␤2␥2 containing receptor observed within the hippocampus and Purkinje cells and the ␣4␤␦ receptor localised in the dentate gyrus. This suggests important regional differences in neuroactive steroid sensitivity that may regulate the action of these steroids in the foetal brain. Furthermore, delta-subunit containing receptors are largely located extra-synaptically on neurones where they provide a tonic level of inhibition rather than modulating the action of GABA within the synapse (phasic activity) [51,52]. Subunit composition most sensitive to neuroactive steroid modulation are associated with extra synaptic sites [48,49] most likely resulting in tonic inhibitory actions [53,54]. The high concentrations of allopregnanolone in the foetal brain, and potentially other progesterone metabolites to some extent, are consistent with a role for these steroids in maintaining a suppressive action on CNS activity. Furthermore, the lipophilic nature of allopregnanolone may result in action on a number of nearby cells promoting a broader suppression. This is supported by findings that inhibition of neuroactive steroid synthesis during pregnancy, either by lowering placental progesterone synthesis, using a 3␤-hydroxysteroid dehydrogenase inhibitor, or blocking the metabolism of progesterone to allopregnanolone,

markedly increases arousal-like behaviour and excitation in the ovine foetus [55,56]. Furthermore, this indicates that allopregnanolone levels markedly influence behavioural states during foetal life and may have a major impact on brain development.

5. Neurosteroid regulation of foetal behavioural state and effects on development The role of GABAA receptors in the regulation of excitability has been found to change with developmental age. Stimulation of GABAA receptor activity is inhibitory during late gestation in the sheep foetus, and GABAA receptor agonists increase inhibitory activity [55]. In early development, limited expression of the K+ /Cl− cotransporter (KCC2) and greater expression of the Na+ KCC1 (NKCC1) transporter in immature neurons has been suggested to lead to increased intracellular Cl− . This would result in GABAA receptor stimulation leading to increased Cl− outward conductance that would tend to depolarize the cells [57] and promote excitation. In rodent species with short gestations the brain is immature at birth and KCC2 expression is low [58]. Thus, GABAA receptor stimulation with agonists such as barbiturates does not reduce, and may even increase, excitation in foetal and early neonatal life in these species. Limited KCC2 expression in human neonates, particularly those born very preterm, has been suggested to explain the poor anti-seizure responses to barbiturates and benzodiazepines in these neonates compared with older infants [59]. In contrast, recent studies of Begestovski and co-workers suggest this may occur to a greater extent in slice preparations where expression of each channel may decline compared to in vivo expression [60]. These authors suggest some of the excitatory effect may be due to artificial changes in transporters in these preparations. This proposal is consistent with our findings in foetal sheep, showing that stimulation of GABAA receptors with neuroactive steroid agonists leads to inhibition of CNS activity from the last third of gestation onwards [61]. Our findings show that the action of allopregnanolone on GABAA receptor regulates foetal CNS activity [56]. Thus, in normal pregnancy, the action of neuroactive steroids is to reduce activity and maintain sleep-like behaviour that perhaps functions to limit cerebral oxygen consumption in a species where gestation is relatively long and the foetus reaches a high degree of maturity by the time of birth. Assessment of foetal activity, specifically breathing and heart rate variability, are key to monitoring the health of the human foetus [62]. In foetal sheep organised patterns of alternating episodes of high voltage (HV) and low voltage (LV) electroencephalographic (EEG) activity are present from at least 120 days of gestation (term ∼147; [63]). These EEG patterns, together with other behavioural measures (eye movements, trunk muscle electromyogram [EMG]), appear to resemble ‘quiet’ and ‘active’ or REM sleep in the adult. This foetal ‘sleep’ is interrupted only briefly by short periods of arousal-like activity, characterised by LV ECoG activity coupled with the simultaneous appearance of vigorous muscle EMG activity, episodes of foetal breathing movements, and the presence of eye activity [27,55,63,64]. Foetal ‘arousal’ accounts for approximately 5% of total time. This low incidence may be attributed to the presence of GABAA receptor-active neuroactive steroids since inhibition of progesterone production [27] or its conversion to 5␣reduced metabolites significantly increases the incidence of this arousal-like activity [55]. Adequate time in active sleep has been shown to contribute to developmental processes and disruption of normal sleep state patterns may be detrimental. The incidence of REM-like sleep falls after birth and this together with the reduction in allopregnanolone levels may contribute to the increased incidence of behavioural problems observed after preterm birth.

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6. The role of neuroactive steroids on brain growth and myelination There is now increasing evidence that neurosteroids improve outcomes following hypoxic/ischaemic brain injury in adults by aiding tissue repair [6]. These processes involve increased production of allopregnanolone and its interaction with GABAA receptors. We have shown that suppression of allopregnanolone production alone will increase apoptotic cell numbers in the foetal brain in the absence of any injury process [65]. Reduced allopregnanolone levels also resulted in an increase in proliferation of astrocytes, whereas oligodendrocytes were reduced in number. These effects of inhibiting neuroactive steroid synthesis were blocked by coinfusion of alfaxalone, suggesting that allopregnanolone in the foetal brain is required to maintain constitutive levels of cell death and proliferation in late development [65]. Previous findings show that neuroactive steroids stimulate myelination [66], an action suggested to involve neuroactive steroid-induced stimulation of GABAA receptors, which appear to indirectly affect oligodendrocytes. Recovery from acute perinatal hypoxic injury involved increased proliferation of oligodendrocyte progenitor cells and their maturation into mature oligodendrocytes [67]. A considerable component of this action has been suggested to involve neuroactive steroid-mediated stimulation of GABAA receptors associated with oligodendrocytes [68]. Stimulation of GABAA receptors has also been reported to block the action of glutamate at AMPA receptors on oligodendrocytes [10], and to stimulate the proliferation of oligodendrocyte progenitor cells [69]. 5␣-Reductase-2 is strongly expressed in oligodendrocytes, which could therefore raise allopregnanolone levels locally and stimulate myelination [35]. Evidence also supports a direct effect on progesterone in stimulating myelination, as assessed by enhanced myelin basic protein expression [70]. This action involves the stimulation of intracellular progesterone receptors that are expressed by these cells, to increase myelin production. In addition, progesterone has also been shown to directly stimulate the maturation of oligodendrocyte progenitor cells [71]. Together these findings suggest progesterone may stimulate oligodendrocytes to increase myelination by two pathways, firstly by the stimulation of progesterone receptors and secondly by conversion to allopregnanolone and allopregnanolone-induced stimulation of GABAA receptors [72]. Studies of guinea pig pregnancies, in which allopregnanolone synthesis was pharmacologically inhibited with the 5␣-reductase inhibitor, finasteride, demonstrated a reduction in myelination in the subcortical white matter, as measured by myelin basic protein (MBP) expression (Fig. 3 [11]). Some caution is needed concerning findings with finasteride which blocks both the conversion of testosterone to 5␣-dihydrotestosterone as well as progesterone to 5␣-dihydroprogesterone and may also raise progesterone concentrations. Despite these considerations, our observations in the guinea pig support the role of allopregnanolone in influencing appropriate levels of myelination during late gestation.

7. Allopregnanolone and the neurotrophin release The observation that allopregnanolone has neurotrophic actions in animal models of degenerative disease, supports a role that involves stimulating oligodendrocyte maturation [4]. Stimulation of GABAA receptors by allopregnanolone has been found to increase the expression of brain derived neurotrophic factor (BDNF) [73]. BDNF is an important mediator of synaptic plasticity and neurite growth. Thus, the protective action of allopregnanolone following neurotrauma may involve stimulation of BDNF production [74,75]. BDNF expression is reduced in the hippocampus following intrauterine growth restriction (IUGR) in guinea pigs

Fig. 3. Effect of finasteride (Fin, hatched bars) or control (closed bars) treatment on (A), allopregnanolone concentrations in foetal brain (control n = 4, finasteride n = 6) and (B) myelin basic protein (MBP) expression (quantified as percentage immunostaining coverage area; control n = 10, finasteride n = 10) in the sub cortical white matter of the foetal guinea pig brain. Data mean ± SEM. *P < 0.05 finasteride vs control [11].

[76]. The effective ‘withdrawal’ of this steroid, particularly in the case of preterm birth, may influence brain development through decreased allopregnanolone levels and indirectly by reduced BDNF production. The effect of an early loss of the placental progesterone or IUGR on brain development and organisation may be complex involving multiple steps in the steroidogenic pathway and interactions with other trophic systems. 8. Protective role of neuroactive steroids following acute injury The suppression of neuroactive steroid levels markedly potentiates cell death in a foetal sheep model of hypoxia/ischaemic injury, whereas replacement and supplementation of levels of allopregnanolone, or co-administration of the allopregnanolone analogue, alfaxalone, blocks this injury [65]. These observations indicate that the synthesis of allopregnanolone protects against hypoxic/ischaemic injury in the late gestation foetus. Furthermore, studies of birth asphyxia in a small precocial animal species, the spiny mouse (Acomys cahirinus), have shown pre-treatment of dams with allopregnanolone protected the hippocampus of the offspring from the decreases in long-term potentiation (LTP) caused by birth asphyxia [77]. These findings indicate that the presence of gestational levels of allopregnanolone creates resistance to damage at birth. Together, recent findings, suggest potential clinical uses of supplementation therapy following adverse events at birth. Such therapies may mimic endogenous processes since allopregnanolone has not only a major role in controlling CNS activity, but processes leading to increased synthesis appear important in protecting the brain. Continuous measurement of allopregnanolone levels in the foetal brain before and after acute hypoxia, induced by occlusion of the umbilical cord, have shown that this insult leads to a marked rise in allopregnanolone levels over the following hours [13]. An occlusion of 10 min causes a severe deterioration in foetal blood gas parameters and lead to a marked rise in allopregnanolone concentrations in the foetal grey and white matter that continues for up to 4 h after the insult [13]. This response is protective against damage since if it is blocked by treatment with finasteride there is markedly elevated hypoxia-induced cell death [12]. Further studies showed that the allopregnanolone response involves upregulation of 5␣-reductase type-2 in the foetal brain [13]. Interestingly a similar increase occurs in the newborn lamb in response

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expression in the placenta. In addition, 5␣-reductase-2 expression tended to be reduced in the brain of IUGR guinea pig foetuses [11]. These finding suggest reduced neurosteroid responses may contribute to the adverse outcomes seen when IUGR foetuses are delivered prematurely. Studies with human placentae show that the expression of both 5␣-reductase isoforms is lower in the placenta from preterm pregnancies (Fig. 2). This suggests there may be a period of reduced exposure to neuroactive steroid prior to preterm birth and this deficiency may be further exacerbated for IUGR. Several studies have evaluated maternal progesterone as a treatment to reduce the risk of preterm labour [84] and further analysis is ongoing [85], however, there is little data on effects on the foetus [86]. The potentially positive effects of increasing neuroactive steroid levels by the use of replacement or supplementation strategies perhaps using progesterone following premature birth, and in IUGR foetuses, respectively, on reducing brain injury require evaluation.

11. Effect of glucocorticoids and stress on neuroactive steroids synthesis Fig. 4. Effects of alfaxalone on electroencephalographic (EEG) spiking activity in foetal sheep. Spiking activity was quantified as high amplitude spikes per 5 min epochs. Time is shown as 5 min epochs beginning after the conclusion of a 10 min hypoxia/ischaemia episode (time 0). Horizontal bar = foetal infusion of alfaxalone (5 mg/kg/min, n = 6, grey bars) or vehicle (black bars, n = 6). Data mean ± SEM. *P < 0.05 alfaxalone vs vehicle [8].

to acute immune stimulation [14]. These observations indicate that allopregnanolone responses to acute stresses represent an endogenous neuroprotective mechanism during the perinatal period. 9. Neuroprotection and suppression abnormal EEG activity Seizure-like activity makes a major contribution to the cell death caused by acute foetal hypoxia [78–80]. Neuroactive steroids modulation of GABAA receptors potently suppresses seizure activity induced by injection of the GABAA receptors antagonist, picrotoxin, in the foetus [56]. Furthermore we have found alfaxalone treatment also reduced the abnormal EEG pattern in foetal sheep following acute hypoxia produced by a brief period of umbilical cord occlusion (Fig. 4) [8]. These findings indicate that a component of the neuroprotective action of allopregnanolone arises from its action in suppressing seizure-like EEG activity. Furthermore, seizure activity in preterm neonates, that can go unnoticed without appropriate EEG monitoring, may result from the premature loss of neuroactive steroid induced suppression. 10. Neurosteroid disruption following growth restricted pregnancies and preterm birth Preterm birth and IUGR are serious compromises of pregnancy that can lead to brain injury [81]. Both conditions may result in reduced neuroactive steroid availability to the foetal brain either by the premature loss of placental precursor support, in the case of preterm birth, or of a reduced capacity for steroid synthesis in the placenta. In many cases of preterm birth, the foetus is also growth restricted which may further reduce neuroactive steroid levels [81]. Growth restriction results from placental insufficiency which limits nutrient supply to the foetus and in turn usually results from inadequate placental growth [82]. In sheep, embolisation of the foetal side of the placenta to induce placental insufficiency causes chronically reduced blood gas parameters and elevated cortisol levels in the foetus and growth restriction [83]. However, in contrast to acute stress, no increase in neuroactive steroid levels was observed after this chronic stress and there was no increase in 5␣-reductase-2

Antenatal corticosteroid treatment with synthetic steroids such as betamethasone is now standard practice to ensure preterm neonates have adequate lung function to survive. While few adverse effects with a single antenatal dose of betamethasone have been reported [87,88], considerable concern remains over negative effects of multiple doses on the brain. Potential effects on neuroactive steroid synthesis should be considered. Animal studies have shown a reduction in brain weight and myelination after repeated betamethasone treatments [89,90]. These effects could involve suppression of neuroactive steroid-dependent stimulation of maturational processes. Repeated betamethasone treatment has been found to reduce the expression of 5␣-reductase-2 in the guinea pig placenta, and to lower 5␣-reductase-2 expression in the male foetal hippocampus suggesting males may be very sensitive to these exposures (Fig. 5 [19]). Multiple betamethasone exposures also decreased GFAP immunostaining in the hippocampus of the male foetuses indicating disrupted levels of astrocyte development (Fig. 5) [19]. These findings, however, suggest that repeated elevated exposure to cortisol in response to stressors may also lower neuroactive steroid concentrations and males may be most vulnerable to the resulting adverse effects. The reason for these effects being restricted to male foetuses is unclear, although, interaction with androgens synthesis or action may be involved. Certain testosterone metabolites may be neuroactive although little is know of the levels of these metabolites in the foetus. Similar effects to those seen with betamethasone were observed when allopregnanolone levels are suppressed by 5␣-reductase inhibition (Fig. 3), suggesting that at least some of the negative effects of synthetic glucocorticoids may result from the suppression of endogenous neuroactive steroid synthesis [19]. Together these observations are further consistent with the possibility that chronically raised glucocorticoid levels, either by exogenous treatment or endogenously following chronic stress, could reduce neuroactive steroids in the brain and delay or disrupt late gestation brain development. The potential for stress-induced rises in glucocorticoid concentrations to influence neuroactive steroid synthesis may be an important mechanism mediating the adverse effects of maternal stress in late pregnancy that is associated with adverse perinatal outcomes. Epidemiological studies show strong associations between prenatal maternal stress and adverse outcomes including those relating to cognitive, behavioural and emotional development of offspring [18,91]. Prenatal stress is also associated with increased incidences of childhood behavioural problems [92–94]. Male foetuses appear more vulnerable demonstrating higher rates

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Fig. 5. Effect of repeated betamethasone (beta, open bars) or vehicle (control, closed bars) treatment of guinea pigs at 65 days of gestation on: (A), 5␣-reductase-2 (5␣-R2) mRNA expression in the placenta from male and female foetuses (combined male and female, control n = 10; beta n = 10); (B) 5␣-reductese-2 (5␣-R2) mRNA expression in the brain of male foetuses (control n = 6; beta n = 6); and (C) glial fibrillary acidic protein (GFAP, quantified as percent immunostained coverage area) in the hippocampus (CA1) of male foetuses (control n = 6; beta n = 6). Data mean ± SEM. *P < 0.05 betamethasone vs control treatment [19].

of learning and memory deficits, and hyperactivity disorders following exposure to late gestation prenatal stress [92,95]. These adverse behavioural outcomes are supported by animal studies showing stress in pregnancy is associated with perturbations in foetal brain development. Stress in late gestation has been found to result in disruptions of myelination and glial cell proliferation occurs over this period [96]. The hippocampus is susceptible to these disruptions and leading to neuropathologies later in life [97]. Excessive exposure of cortisol in response to stress and the subsequent reduction in normal neuroactive steroid exposure may contribute the deleterious effects of prenatal on foetal brain development. Prenatal stress raises maternal cortisol levels and while placental 11␤-hydroxysteroid dehydrogenase type-2 does limit the passage of maternal cortisol to the foetus, this barrier is not complete and foetal cortisol levels rise when maternal levels are elevated [98]. Allopregnanolone treatment during late gestation has been found to reduce the deleterious effects of prenatal stress on rat pups [99], supporting a link between reduced allopregnanolone levels and adverse developmental outcomes. Thus neuroactive steroid supplementation treatment may have value in reversing the potentially negative effects of stress-induced elevations in cortisol in the foetus in these pregnancies. Placental 11␤-hydroxysteroid dehydrogenase type-2 converts cortisol to cortisone which has low activity at the glucocorticoid receptors. This action protects the foetus from high maternal circulating cortisol levels in response to acute stress. Thus foetal cortisone levels will be increased after cortisol levels rise in the maternal circulation. The relative importance of elevated levels of cortisol or cortisone concentrations in response stress on neuroactive steroid pathways in the foetus is unclear. Several cortisol and cortisone metabolites are potential agonists at the GABAA receptor. These steroids include 5␣,3␣-reduced metabolites of cortisol, tetrahydrocortisol (5␣TH-cortisol), and cortisone, tetrahydrocortisone (5␣TH-cortisone) which have been reported to be present in concentrations that would modulate GABAergic pathways [20]. In addition, other metabolites of cortisone that are immediate precursors of these steroids are released by the placenta following exposure to high cortisol levels [100]. Conjugates of these cortisol metabolites are also found in urine of preterm neonates [101]. These observations suggest the potential risk of increased of glucocorticoid-induced suppression of allopregnanolone production may be negated by the increased levels of cortisol and cortisone metabolites. However, little is known about the effects of these metabolites either in the foetus or neonate.

12. Neuroactive steroid responses to stress in the neonatal period The concentrations of both progesterone and allopregnanolone drop rapidly in the brain and plasma with the loss of the placenta

after birth. While both 5␣-reductases and P450cssc expression in the brain continue without decline after birth the dramatic drop in neuroactive steroid levels is consistent with the placenta supporting gestational neurosteroid concentrations [2]. The postpartum fall in neuroactive steroid concentrations in the maternal circulation has been connected with depressive illness after birth, however, such a relationship is more complex as neuroactive steroids decline rapidly after birth whereas these conditions appear to develop more slowly [102,103]. Similarly in the neonate, multiple changes, such as altered GABAA receptor subunit expression may heighten the effect of steroid withdrawal and further increase excitability in the days after delivery [104]. Despite the decline in baseline allopregnanolone concentration in the brain after birth, as with the foetus, neuroactive steroids do protect the neonate under adverse conditions [14]. Studies in neonatal sheep have shown a marked increase in allopregnanolone concentrations in the brain following an infectious challenge induced by injection of lipopolysaccharide. This response markedly suppressed excitability and raised seizure threshold [105]. Whether such changes are seen in premature neonates has not been investigated, but should the responses be less robust there may be greater sensitivity to excess excitation. Further studies of neuroactive steroid responses to stressors in neonates delivered at increasing degrees of immaturity are required to assess the potential value of replacement strategies after preterm birth.

13. Conclusions Allopregnanolone and potentially other progesterone metabolites yet to be measured act on specific GABAA receptors to suppress excitation during late gestation [55,56]. The production of progesterone by the placenta leads to relatively high concentrations of allopregnanolone in the foetal plasma and brain. These concentrations may be the highest levels attained throughout life and have a major role in modulating excitability in the foetus and protecting the foetal brain from excitotoxicity [12]. The enzymes required for the synthesis of allopregnanolone including 5␣-reductase-2 are present in the placenta and foetal brain during late gestation and may regulate allopregnanolone levels, however, progesterone production by the placenta is required to maintain gestational allopregnanolone levels. Concentrations fall dramatically after removal of the placenta at birth [2]. In this way the placenta exerts a major influence over the foetal brain thereby providing trophic support. Gestational allopregnanolone concentrations protect from hypoxic/ischaemic events and associated seizures that are exacerbated if allopregnanolone levels are suppressed. In addition allopregnanolone supports late gestation brain development since suppression of allopregnanolone synthesis causes disruption of normal levels of apoptosis and may reduce myelination [65].

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