Neuroactive steroids attenuate oxytocin stress responses in late pregnancy

Neuroscience 138 (2006) 879 – 889

NEUROACTIVE STEROIDS ATTENUATE OXYTOCIN STRESS RESPONSES IN LATE PREGNANCY J. A. RUSSELL* AND P. J. BRUNTON

Oxytocin is produced by magnocellular neurones in the supraoptic and paraventricular nuclei, is released predominantly from the axon terminals in the posterior pituitary, and its indispensable role is in driving the milk ejection reflex during suckling in lactation, by stimulating myo-epithelial cell contractions in the mammary gland. Oxytocin also has a major role in stimulating contractions of the myometrium during parturition, particularly in the final stages of birth (Russell et al., 2003). In the rat, oxytocin is secreted in response to hyperosmotic disturbance, and causes natriuresis, via stimulation of atrial natriuretic peptide secretion (Gutkowska et al., 1997). Oxytocin is also released in the median eminence from swellings of axons en passage to the posterior pituitary, and in the rat, oxytocin can act like vasopressin via the V1b receptors on corticotrophs to add to the stimulatory action of corticotrophin-releasing factor on ACTH secretion (Ma et al., 2005).

Laboratory of Neuroendocrinology, Centre for Integrative Physiology, School of Biomedical Sciences, College of Medicine and Veterinary Medicine, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK

Abstract—In late pregnant rats neuroendocrine stress responses, expressed as increased oxytocin secretion and activation of the hypothalamo–pituitary–adrenal axis, are attenuated. These adaptations preserve the oxytocin store for parturition and prevent pre-term birth, and protect the fetuses from adverse programming by exposure to excess glucocorticoid. Mechanisms of adaptations for oxytocin neurones are reviewed, using challenge with systemic interleukin-1␤, simulating activation of immune signaling by infection, as a stressor of special relevance in pregnancy. In virgin rats, systemic interleukin-1␤ stimulates the firing of oxytocin neurones, and hence oxytocin secretion, but interleukin-1␤ has no effects in late pregnant rats. This lack of response is reversed by naloxone treatment just before interleukin-1␤ administration, indicating endogenous opioid suppression of oxytocin responses in late pregnancy. This opioid presynaptically inhibits noradrenergic terminals impinging on oxytocin neurones. Finasteride pretreatment, inhibiting progesterone conversion to allopregnanolone, a positive GABAA receptor allosteric modifier, also restores an oxytocin response to interleukin-1␤. This finasteride effect is reversed by allopregnanolone treatment. In virgin rats allopregnanolone attenuates the oxytocin response to interleukin-1␤, which is exaggerated by naloxone. The effects of naloxone and finasteride in late pregnant rats in restoring an oxytocin response to interleukin-1␤ are not additive. Accordingly, allopregnanolone may both enhance GABA inhibition of oxytocin neurone responses to interleukin-1␤, and induce opioid suppression of noradrenaline release onto oxytocin neurones. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved.

Stimulation of neuroendocrine stress mechanisms by interleukin-1␤ Stressors that stimulate oxytocin secretion include emotional stressors such as forced swimming (Neumann et al., 1998), immobilization (Williams et al., 1985), social defeat (Neumann et al., 2001), and physical stressors, including cholecystokinin (Douglas et al., 1995) and interleukin-1␤, a cytokine produced by activated immune cells, particularly macrophages (Buller et al., 2001a). Cholecystokinin and interleukin-1␤ act via vagal afferents, after i.p. injection (Renaud et al., 1987; Fleshner et al., 1995), and locally in the nucleus tractus solitarius (NTS) to activate noradrenergic (A2) neurones, which project directly to magnocellular oxytocin neurones in the paraventricular nucleus and supraoptic nucleus, and to corticotrophin-releasing factor neurones in the parvocellular paraventricular nucleus, exciting both types of neurone (Day and Sibbald, 1988; Sun and Ferguson, 1997; Ericsson et al., 1994; Buller et al., 1998). A1 neurones in the ventrolateral medulla have a less important role in mediating interleukin-1␤ actions on oxytocin and corticotrophin-releasing factor neurones (Ericsson et al., 1994; Buller et al., 2001a). After i.v. administration, the predominant site of interleukin-1␤ action is through interleukin receptors on the endothelial cells of small blood vessels supplying the noradrenergic nuclei (Ericsson et al., 1997). Cyclo-oxygenase 2 is activated, and the locally generated prostaglandin E2 diffuses into the brain parenchyma, exciting NTS noradrenergic (A2) neurones via prostaglandin E2 EP4 G-protein-coupled receptors (Buller et al., 1998; Zhang and Rivest, 1999; Rivest, 2001). Interleukin-1␤ also acts directly on the posterior

Key words: allopregnanolone, finasteride, hypothalamo–pituitary–adrenal axis, interleukin-1␤, opioids, progesterone.

Stress increases secretion of hypothalamo–pituitary–adrenal axis hormones, but in rats also increases oxytocin secretion, particularly in females (Williams et al., 1985). This paper reviews evidence that these responses are attenuated in late pregnancy, the significance of the attenuation, and the mechanisms involved, focusing on responses to immune challenge and oxytocin neurone adaptations. *Corresponding author. Tel: ⫹44-0-131-650-2861; fax: ⫹44-0-131-6502872. E-mail address: [email protected] (J. A. Russell). Abbreviations: allopregnanolone, 3␣-hydroxy-5␣-pregnan-20-one; NTS, nucleus tractus solitarius.

0306-4522/06$30.00⫹0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2005.09.009

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1994; Buller et al., 2001a). Within the paraventricular nucleus noradrenaline interacts with glutamate mechanisms within the immediate vicinity of the oxytocin and corticotrophin-releasing factor neurones (Daftary et al., 1998), and descending projections from the paraventricular nucleus positively modulate the responses of NTS neurones to systemic interleukin-1␤ (Buller, 2003). We have used this well-defined model of activation of the magnocellular oxytocin system by systemic (i.v.) interleukin-1␤ to study plasticity of stress responses in late pregnancy, and the roles of endogenous opioids and sex steroid hormones or their neuroactive products. This extends previous studies on adaptations in responses to emotional stressors (Douglas et al., 1998; Neumann et al., 1998).

pituitary, modestly enhancing (by about 20%) electricallystimulated oxytocin secretion (Christensen et al., 1990). Not all NTS neurones excited by interleukin-1␤ are noradrenergic (Ericsson et al., 1994), but noradrenergic neurones are most important in mediating stimulation of oxytocin and corticotrophin-releasing factor neurones. In vivo microdialysis shows increased release of noradrenaline in the paraventricular nucleus following interleukin-1␤ (Brunton et al., 2005). NTS catecholaminergic neurones are activated (express Fos) after systemic interleukin-1␤, including neurones retrogradely labeled by tracer injected into the paraventricular nucleus (Ericsson et al., 1994). Neurotoxic lesion of NTS neurones, either with local ibotenic acid or with 6-hydroxydopamine injections into the paraventricular nucleus or supraoptic nucleus, greatly reduces activation of oxytocin or corticotrophin-releasing factor neurones by systemic interleukin-1␤ (Buller et al., 2001a). Neurones of the central nucleus of the amygdala receive an A2 projection directly or indirectly, via the parabrachial nucleus, and are activated by systemic interleukin-1␤; these amygdala neurones project via the bed nucleus of stria terminalis to regulate corticotrophin-releasing factor neurones (Buller et al., 2001b, 2004; Crane et al., 2003). However, this indirect pathway is not as important as the direct noradrenergic projection to corticotrophinreleasing factor and oxytocin neurones (Ericsson et al.,

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Convergence of stimuli: parturition, cholecystokinin, interleukin-1␤ The brainstem noradrenergic input to oxytocin neurones is importantly involved in mediating positive feedback stimulation of oxytocin secretion by neural signals from the uterus and birth canal during parturition. Thus, noradrenergic NTS neurones, identified as projecting to the supraoptic nucleus by retrograde labeling, express Fos during parturition (Meddle et al., 2000). Furthermore, noradrenaline release in the supraoptic nucleus is increased

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Fig. 1. Stress and oxytocin neurones: GABA, interleukin-1␤ and naloxone. (a, b) Oxytocin was radioimmunoassayed in plasma samples from a jugular cannula implanted (halothane anesthesia) 5 days before experiment (pregnancy day 21 [day 1: vaginal plug], 1–2 days before expected parturition). Data are fold increases over basal group mean (e.g. in a): basal values, virgin and late pregnant, [n⫽13 and 18]: 2.5⫾0.1 and 2.5⫾0.03 pg/ml n.s); dashed line⫽no change. Numbers of rats are in the bars. Statistics: RM ANOVA and Student-Neuman-Keuls tests performed on raw data (not shown). (a) Oxytocin secretion was increased 30 min after i.p. picrotoxin (GABAA antagonist; 7.5 mg/kg, i.p., a non-ictal dose; P⬍0.05), with no difference between late pregnant and virgin rats (unpublished observations with S. Ma). (b) Interleukin-1␤ (IL-1␤, 0.5 ␮g/kg, i.v.) increased oxytocin secretion in conscious virgin, but not in late pregnant rats, except after naloxone (5 mg/kg i.v. 15 min before interleukin-1␤). * P⬍0.05 vs other groups. (c) Individual supraoptic neurones were recorded extracellularly in urethane-anesthetized rats. Interleukin-1␤ excited oxytocin neurones (identified by firing pattern and excitation by i.v. cholecystokinin) only in virgin rats (* P⬍0.05); data are mean changes between 5 min before and 10 –15 min after interleukin-1␤. Given 10 min after naloxone, interleukin-1␤ was excitatory in late pregnant rats, but less effective in virgin rats (# P⬍0.05 vs vehicle). (Brunton et al., 2004b).

J. A. Russell and P. J. Brunton / Neuroscience 138 (2006) 879 – 889

during natural or simulated parturition, and central administration of an ␣-1 receptor antagonist reduces Fos expression in supraoptic neurones (Herbison et al., 1997; Douglas et al., 2001). Electrophysiological studies show that systemic cholecystokinin and stimulation of the birth canal activate different sets of NTS neurones (Bailey and Wakerley, 1997); whether interleukin-1␤ and afferents from the birth canal converge onto the same set of NTS neurones has not been studied. Nonetheless, it seems from several studies that the NTS noradrenergic neurones excited by these different stimuli converge to stimulate essentially all of the oxytocin neurones (e.g. Fig. 1c; Renaud et al., 1987; Brown et al., 1996; Douglas et al., 2001). However stimulated, increased secretion of oxytocin from the posterior pituitary near the end of pregnancy may stimulate uterine contractions and pre-term labor, provided oxytocin receptor expression is up-regulated, and that luteolysis has removed the progesterone block to oxytocin action on the myometrium (Antonijevic et al., 2000). Human studies indicate that uterine infection is a factor in pre-term labor, in which oxytocin is important (Goldenberg and Culhane, 2003). In animal models, intra-uterine administration of lipopolysaccharide (LPS), or inactivated Escherichia coli, can cause pre-term delivery in rats, or more reliably in mice (Elovitz and Mrinalini, 2004), but it is not known whether oxytocin secretion is stimulated in these models of pre-term birth triggered by intra-uterine infection and inflammation. Mechanisms of attenuated oxytocin stress responses in late pregnancy The studies reviewed here indicate that prevention of premature activation of oxytocin secretion by stressors in late pregnancy, and by systemic interleukin-1␤ in particular (Fig. 1; Douglas et al., 1998; Neumann et al., 1998; Brunton et al., 2004b), involves central endogenous opioid mechanisms that are activated in pregnancy, and neuroactive steroid produced by metabolism of progesterone. Possible mechanisms for the attenuation of oxytocin neurone stress responses in pregnancy a priori include: reduced capacity of the system to respond, reduced effectiveness of excitatory input, and increased effectiveness of inhibitory input. Response capacity of the magnocellular oxytocin system The posterior pituitary store of oxytocin is increased by about a third at the end of pregnancy, mainly as a result of decreased secretion, providing for the large stimulated increase in oxytocin secretion during parturition (Russell et al., 2003). Despite this, stressors generally less effectively stimulate oxytocin secretion in late pregnancy (Douglas et al., 1998; Neumann et al., 1998; Brunton et al., 2004b); systemic interleukin-1␤ increases oxytocin secretion ⬎three-fold in virgin rats, but has no significant effect in late pregnant rats (Brunton et al., 2004b; Fig. 1b). The attenuated oxytocin response to stressors in pregnancy is a result of reduced central drive to oxytocin neurones, since picrotoxin (a GABAA receptor antagonist, which will

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interrupt the tonic inhibition of oxytocin neurones by their extensive GABA input (as discussed further below) stimulates the secretion of similar large amounts of oxytocin in virgin and late pregnant rats (Fig. 1a). Moreover, normal oxytocin responses are seen after blocking endogenous opioid or neurosteroid mechanisms as discussed below. The lack of an oxytocin response is not attributable to inhibition by corticosterone, as suggested by other findings (Harbuz et al., 1996), because interleukin-1␤ does not activate the hypothalamo–pituitary–adrenal axis in late pregnant rats (Brunton et al., 2005). This lack of a hypothalamo–pituitary–adrenal response to stressors in pregnancy is also not due to lack of secretory capacity (Ma et al., 2005). Modified neural input Oxytocin responses in late pregnancy to forced swimming, or to other emotional stressors are reduced (Douglas et al., 1998; Neumann et al., 1998), which contrasts with complete suppression of these responses to systemic interleukin-1␤ (Fig. 1b). This can be attributed to differential inhibition in late pregnancy of the different circuits that process these emotional and physical stressors. The former involve primarily limbic circuits (Herman et al., 2003), and the latter input from the brainstem, as discussed above. Nonetheless, common mechanisms, involving inhibition by endogenous opioids and neuroactive steroid metabolites of progesterone can account predominantly for the attenuated or suppressed oxytocin responses to stressors in late pregnancy. Specifically, the results might be explained by endogenous opioids predominantly acting by suppressing noradrenaline release onto the oxytocin neurones, and by progesterone metabolites enhancing GABA inhibition. The evidence for these inhibitory mechanisms is reviewed next, with focus on responses to systemic interleukin-1␤, because the strong oxytocin secretory responses to interleukin-1␤ seen in virgin rats are essentially completely suppressed in late pregnancy, and the central neural pathway mediating actions of interleukin-1␤ on oxytocin, and corticotrophin-releasing factor, neurones is clearly characterized. Decreased excitatory coupling Fos immunocytochemistry shows similar numbers of NTS neurones are activated 90 min after systemic interleukin-1␤ in virgin and late pregnant rats (increasing from basal levels of ca. 70 to ca. 190 and 180 Fos-positive neurones/mm2, respectively; Brunton et al., 2005). As ca. 42% of NTS neurones are catecholaminergic (Ericsson et al., 1994), it follows that the number of NTS noradrenergic neurones activated by systemic interleukin-1␤ is not significantly reduced in late pregnancy. Thus the interleukin-1␤ transduction mechanisms in the medulla are functional in pregnancy. Nonetheless, whereas basal noradrenaline release in the paraventricular nucleus, measured with microdialysis and electrochemical detection, is similar in virgin and late pregnant rats, systemic interleukin-1␤ increases noradrenaline release 2.5-fold within 15 min in virgin rats, but there is no increase in late pregnancy (Brunton et al., 2005).

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This may explain the failure of systemic interleukin-1␤ administration to increase oxytocin secretion in late pregnancy. As the cell-bodies in the NTS are activated by systemic interleukin-1␤, as shown by Fos expression, the failure of these neurones to release noradrenaline in the paraventricular nucleus is likely a result of pre-synaptic inhibition. This could be through up-regulated autoinhibitory adrenergic ␣2-receptors, or via auto-inhibition by a co-produced opioid peptide, as explored below. Reduced expression of adrenergic ␣1A-receptor subunit mRNA in the magnocellular paraventricular nucleus in late pregnancy (Douglas, 2005; Douglas et al., 2005), does not explain suppressed oxytocin neurone responses to systemic interleukin-1␤. This is because noradrenaline release and oxytocin neurone responses after interleukin-1␤ in late pregnancy are restored within a few minutes after treatment with naloxone, an opioid receptor antagonist (Brunton et al., 2004b; Fig. 1b, c). It also follows that lack of noradrenaline release after interleukin-1␤ in late pregnancy is not due to lack of capacity of the brainstem A2 neurones to produce noradrenaline. Role of endogenous opioids. Oxytocin neurones become dominated by endogenous opioid inhibition in pregnancy, and this restrains oxytocin neurone excitation by various stimuli, including during parturition (Leng et al., 1988; Douglas et al., 1995). Thus naloxone increases the stimulated activity of oxytocin neurones in late pregnancy, and restores oxytocin electrical and secretory responses to interleukin-1␤ (Fig. 1b, c). Without pregnancy naloxone has little effect on the oxytocin response to interleukin-1␤, although naloxone can increase stimulated oxytocin secretion by antagonizing actions of ␬-opioids, produced by the oxytocin neurones, on the axon terminals in the posterior pituitary (Leng et al., 1994). Opioid actions at this site are down-regulated in pregnancy (Douglas et al., 1993), so endogenous opioid actions on oxytocin neurones in late pregnancy are deduced to be exerted centrally. Accordingly, naloxone increases Fos expression in oxytocin neurones, and increases somato-dendritic release of oxytocin, in late pregnant but not in virgin rats (Douglas et al., 1995). Furthermore, naloxone increases the firing-rate response of oxytocin neurones to systemic cholecystokinin in late pregnant but not in virgin rats, and naloxone alone has no effect (Douglas et al., 1995). Strikingly, while there is a sustained firing-rate response of oxytocin neurones to i.v. injection of interleukin-1␤ in virgin rats, there is no response at all in late pregnant rats (Brunton et al., 2004b; Fig. 1c). However, i.v. naloxone given 10 min before interleukin-1␤ in late pregnant rats allows a firing-rate response similar to that after interleukin-1␤ in virgin rats (Fig. 1c). In contrast naloxone modestly reduced oxytocin neurone excitation by interleukin-1␤ in virgin rats (Fig. 1c). Together, the oxytocin secretion and electrophysiological data (Fig. 1) indicate that in virgin rats central actions of endogenous opioids have a net, small, facilitatory action on the stimulation of oxytocin neurones by interleukin-1␤, but in late preg-

nancy the predominant action of endogenous opioid is reversed, and is strongly inhibitory. Whereas the posterior pituitary opioid mechanism in virgin rats cannot be selective for the modality of stimulation, the central opioid mechanism that emerges in pregnancy restrains stimulation by signals mediated by brainstem noradrenergic input. These are stimuli arising from the birth canal during parturition, systemic cholecystokinin and interleukin-1␤, but not, for example, from the lamina terminalis input, which mediates osmoregulatory control of oxytocin neurones (Leng et al., 1988; Douglas et al., 1995; Bull et al., 1994). This central opioid mechanism involves ␮-opioid receptors (Leng et al., 1997), and these are evidently on noradrenergic terminals in the magnocellular nuclei (Kutlu et al., 2004). Source of central endogenous opioid. There is increased activity of arcuate nucleus pro-opiomelanocortin neurones in late pregnancy, including an increased density of immunoreactive ␤-endorphin nerve fibers in the supraoptic nucleus (Douglas et al., 2002). However, arcuate neurones are activated by interleukin-1␤ only after a long delay (Brady et al., 1994), and the central opioid restraint in pregnancy is targeted selectively onto specific inputs to oxytocin neurones, as described above. Alternatively, opioid provided by the noradrenergic input would automatically ensure immediate selective presynaptic targeting of this input whenever it is stimulated. This mechanism is indicated by increased basal noradrenaline release in the magnocellular nuclei, and oxytocin secretion, after i.c.v. injection of a selective ␮-opioid antagonist in late pregnant rats (Kutlu et al., 2004). The presence of ␮-opioid receptors on noradrenergic terminals is shown in non-pregnant rats by the suppression by local morphine application, of noradrenaline release in the supraoptic nucleus stimulated by systemic cholecystokinin (Onaka et al., 1995). Indeed, the expression of proenkephalin-A and ␮-opioid receptor mRNAs in the NTS is increased in late pregnancy (by 30% and 40% vs. virgin rats, respectively; Brunton et al., 2005), which suggests increased production and transport of enkephalins and ␮-opioid receptor to the noradrenergic terminals in projection sites. Thus stimulation of the NTS A2 neurones by systemic interleukin-1␤ in late pregnancy is expected to release enkephalins, to act presynaptically and inhibit noradrenaline release. Evidence for this mechanism comes from finding that application of naloxone to the paraventricular nucleus by retromicrodialysis permits a significant increase in noradrenaline release (by 1.7-fold) after systemic interleukin-1␤ in late pregnant rats (Brunton et al., 2005). The involvement of the female sex steroid hormones, and metabolites, in inducing the central opioid inhibitory restraint of oxytocin neurones is discussed below. Increased inhibitory coupling GABA input and neuroactive steroids. The studies with picrotoxin (Fig. 1a) substantiate the importance of tonic GABA inhibition in both the local and remote regulation of oxytocin neurone activity (Leng et al., 2001). The

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effectiveness of the GABA input to oxytocin neurones, arising partly from GABA neurones close to the paraventricular and supraoptic nuclei, is enhanced in late pregnancy by an increased density of GABA synapses on oxytocin neurones (Montagnese et al., 1987), and by the potentiation of GABA action via GABAA receptors. In vitro electrophysiological analysis of GABA actions on oxytocin neurones, from virgin or late pregnant rats, has shown that 3␣-hydroxy-5␣-pregnan-20-one (allopregnanolone) prolongs the opening time of the activated GABAA receptor Cl⫺ channel (Brussaard and Herbison, 2000). The brain content of allopregnanolone is at effective levels in late pregnancy as a result of the increasing secretion of progesterone, by the corpora lutea in the rat, which is metabolized in the brain via 5␣-reductase to 5␣-dihydroprogesterone, and then converted by 3␣-hydroxysteroid dehydrogenase to allopregnanolone (Concas et al., 1998). The cells expressing the converting enzymes in the environment of the oxytocin neurones have not been clearly defined, but in the whole brain, both enzymes are expressed in astroglia and only 5␣-reductase is active in neurones (Melcangi et al., 1993). Changes in GABAA receptor composition in the brain in pregnancy, induced by progesterone or by allopregnanolone (Follesa et al., 2004), and in the supraoptic nucleus in particular (Fenelon and Herbison, 2000), have been considered important in determining effectiveness of allopregnanolone on oxytocin neurones (Brussaard and Herbison, 2000). However, it now seems that local somato-dendritic release of oxytocin opposes, via protein kinase C, allopregnanolone modulation of the GABAA receptor (Koksma et al., 2003), so the relative local concentrations of oxytocin (low in pregnancy, high in parturition; Neumann et al., 1994) and allopregnanolone (high in pregnancy, low at term; Concas et al., 1998), rather than GABAA receptor subunit composition (Lambert et al., 2003), will dramatically alter the effectiveness of GABA action on oxytocin neurones. The enzymes converting progesterone to allopregnanolone are expressed in the medulla (Khanna et al., 1995; Li et al., 1997), so locally produced allopregnanolone may act on NTS neurones projecting to the oxytocin neurones in pregnancy. With luteolysis the withdrawal of progesterone leads to the initiation of parturition a day or so later (Antonijevic et al., 2000), and to reduced allopregnanolone production in the brain (Concas et al., 1998). The time course of the decrease in brain allopregnanolone content near the end of pregnancy is not known in fine detail, but a peak has been measured on day 19 (equivalent to day 20 in our studies; cortex content is ⬎3⫻ level in virgins), and 2 days later (expected term) content is decreased to below the virgin level (Concas et al., 1998). Notably, the brain level of allopregnanolone is only 1.7⫻ the virgin level until at least day 15 of pregnancy, contrasting with the 18⫻ increase in progesterone level at this time (Concas et al., 1998). It is not known whether the activity of the synthesizing enzymes in the brain is up-regulated in pregnancy; in the hypothalamus in lactation the activities of 5␣reductase and 3␣-hydroxysteroid dehydrogenase are respectively decreased and increased (Karavolas and Hodges,

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1993). In pregnancy the effectiveness of the GABA input onto oxytocin neurones is expected to be greatly enhanced by the increased allopregnanolone content of the brain until close to the end of pregnancy. While the focus here is on allopregnanolone, other progesterone-related steroids are produced in increased amounts in pregnancy (Concas et al., 1998), and these can have actions on excitatory amino acid (e.g. NMDA) or GABAA receptors that may oppose the actions of allopregnanolone (Park-Chung et al., 1999). Nonetheless, the next experiments show the predominance of allopregnanolone actions in pregnancy. Allopregnanolone and oxytocin responses to interleukin-1␤. The importance of allopregnanolone production in restraining oxytocin responses to stimulation in late pregnancy has been tested by inhibiting 5␣-reductase activity (with finasteride), and by administration of allopregnanolone or progesterone, its precursor. Late pregnant rats treated with finasteride 20 and 2 h before bloodsampling (to reduce brain allopregnanolone content by up to 90%, at least to the virgin level; Concas et al., 1998), showed a significant oxytocin secretory response to challenge with i.v. interleukin-1␤, in contrast with no response in control late pregnant rats given vehicle and interleukin-1␤ (Fig. 2a). Finasteride had no effect on the oxytocin response to interleukin-1␤ in virgin rats (Fig. 2a). Treatment of late pregnant rats with allopregnanolone together with finasteride completely suppressed the oxytocin response to interleukin-1␤, indicating that finasteride alone acts by preventing allopregnanolone production (Fig. 2b). Furthermore, allopregnanolone treatment (20 and 2 h before experiment) of virgin rats substantially reduced the oxytocin secretory response to challenge with i.v. interleukin-1␤ (Fig. 2c), but similar short-term treatment with progesterone did not significantly reduce the response (Fig. 2d). These findings suggest that in virgin rats the GABAA receptors on oxytocin neurones (or their inputs mediating stimulation by interleukin-1␤) are sensitive to modulation by allopregnanolone, but that the enzyme(s) that metabolize progesterone are not sufficiently active to generate effective brain concentrations of allopregnanolone without pregnancy. The activity of 3␣-hydroxysteroid dehydrogenase is regulated by estrogen, and expression of the mRNA for this enzyme, in the hippocampus, is positively regulated in females by estrogen but not by progesterone (Penning et al., 1985; Mitev et al., 2003). In virgin rats implanted with 17␤-estradiol and progesterone in s.c. capsules for 17 days, to simulate exposure to the pattern, duration and levels of these steroids in pregnancy, the increment in plasma oxytocin concentration 5 min after forced swimming was slightly less than the modest increase in controls with sham implants, although the fold increase in oxytocin secretion was not affected (Fig. 3a; Douglas et al., 2000). A similar experiment to study responses to interleukin-1␤ has not been reported. Steroids and opioids Neuroactive steroid-endogenous opioid interactions (Fig. 4). A key finding from the above experiment came

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Fig. 2. Interleukin-1␤ and oxytocin secretion: effects of finasteride, allopregnanolone (AP) and progesterone (Prog). (a) Finasteride (Fin, 5␣-reductase inhibitor; 25 mg/kg, s.c.) or vehicle (Veh; oil, 0.5 ml/kg) was given 20 and 2 h before interleukin-1␤. Fin restored an oxytocin response to interleukin-1␤ (⫹5 min) in late pregnant rats, without effect in virgins (* P⬍0.05 vs all). (b) AP (3, 1 mg/kg) given with Fin to late pregnant rats suppressed the oxytocin response to interleukin-1␤ (* P⬍0.05 vs Fin). (c) AP (3 and 1 mg/kg) given to virgin rats suppressed the oxytocin response to interleukin-1␤ (* P⬍0.05), (d) Prog (5 and 1 mg/kg) had no significant effect. Fold increases were calculated as in Fig. 1, with statistical comparisons performed on raw data (not shown).

treated virgins, the increase was much greater after naloxone (Fig. 3a). As chronic 17␤-estradiol treatment alone does not produce this effect of naloxone (Douglas et al., 2000), it seems that chronic progesterone exposure (albeit

from studying effects of antagonizing endogenous opioid actions. When naloxone was given before forced swimming, the increase in plasma oxytocin concentration in control virgins was not greater, but in the sex steroid-

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Fig. 3. Swim stress, interleukin-1␤ and oxytocin secretion: opioid and neurosteroid interactions. (a) Virgin rats with 17␤ estradiol and progesterone (E/P) or vehicle implants for 17 days forced to swim for 90 s after i.v. vehicle (Veh) or naloxone (Nal; 5 mg/kg). Data are fold increases in oxytocin concentration from basal, 5 min after swimming. All increases are significant; * P⬍0.05 vs other groups (adapted from Douglas et al., 2000). (b) Virgin rats were given allopregnanolone (AP) or vehicle twice before interleukin-1␤, as in Fig. 2c, and Nal (5 mg/kg) or vehicle 30 min before interleukin-1␤. AP suppression of the oxytocin response to interleukin-1␤ (* P⬍0.05) was reversed by Nal, which amplified the response (# P⬍0.05). (c) Late pregnant rats were given finasteride (Fin) twice before Nal and interleukin-1␤, as in (b). Nal did not further increase the oxytocin response. Fold increases were calculated as in Fig. 1, with statistical comparisons performed on raw data (not shown).

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in the presence of estrogen) may up-regulate opioid inhibition of oxytocin neurone responses to stressors. Supposing that progesterone is active after metabolism to allopregnanolone, we found that, in virgin rats, the suppression by short-term treatment with allopregnanolone of the oxytocin response to interleukin-1␤ is reversed by naloxone. Naloxone not only reversed the attenuation of the oxytocin response caused by allopregnanolone, but it amplified the oxytocin response to interleukin-1␤ (Fig. 3b). Moreover, in late pregnant rats, the restored oxytocin response to interleukin-1␤ by short-term blockade of progesterone conversion to allopregnanolone with finasteride was not further enhanced by naloxone (Fig. 3c). This lack of interaction or summation of naloxone and finasteride effects in increasing the oxytocin secretory response to interleukin-1␤ in late pregnant rats is consistent with induction by allopregnanolone of endogenous opioid suppression of oxytocin neurone excitation by interleukin-1␤ in late pregnancy. The enhanced oxytocin response to swimming or interleukin-1␤ after chronic 17␤-estradiol and progesterone, or short-term allopregnanolone treatment and naloxone (Fig. 3a, b), might be due to increased oxytocin production and storage. However, oxytocin neurones do not express classical progesterone receptors, even in pregnancy (Francis et al., 2002), and they express estrogen receptor ␤, but not -␣ (Somponpun et al., 2004). It is controversial whether oxytocin gene expression increases in pregnancy when estrogen and progesterone production are greatly increased (see Russell et al., 2003). Indeed, progesterone or allopregnanolone restrains oxytocin production (Blyth et al., 2000), and progesterone withdrawal is not necessary for the enhanced response to swimming after naloxone (Douglas et al., 2000). There may be a change in releasability of oxytocin, or a greater firing rate response of oxytocin neurones to interleukin-1␤ after allopregnanolone and naloxone, though this was not seen in late pregnancy after naloxone and interleukin-1␤ (Fig. 1c). Allopregnanolone and opioid expression. Allopregnanolone might up-regulate opioid action on oxytocin release in the posterior pituitary, in which case oxytocin responses to all stimuli would be exaggerated after naloxone. However, chronic 17␤-estradiol and progesterone treatment of virgin rats does not enhance the oxytocin response to hyperosmotic stimulation and naloxone (Bull et al., 1996). Moreover, allopregnanolone does not modulate oxytocin secretion by direct action on the posterior pituitary (Widmer et al., 2003). Hence, it seems likely that allopregnanolone activates a central opioid mechanism. Sex steroid actions on expression of opioid genes: proenkephalin-A. The proenkephalin-A gene has an estrogen response element (Zhu and Pfaff, 1995) and 17␤estradiol treatment rapidly (within 1 h) stimulates proenkephalin-A mRNA expression in the ventromedial nucleus, and this is sustained by progesterone (Romano et al., 1989). NTS A2 neurones projecting to the supraoptic nucleus do not express estrogen receptor-␣ (Voisin et al., 1997), few NTS A2 neurones express estrogen receptor-␤

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(Curran-Rauhut and Petersen, 2003), and very few express progesterone receptor at the end of pregnancy (Francis et al., 2002). So it seems unlikely that estrogen and progesterone act on NTS neurones to increase expression of opioid genes. However, the effect of allopregnanolone treatment of virgin rats in inducing inhibitory opioid tone on oxytocin responses to interleukin-1␤ is quite rapid (⬍20 h), consistent with rapid changes in proenkephalin-A mRNA expression in other circumstances and locations. Proenkephalin-A mRNA expression in the NTS is increased rapidly after interleukin-1␤ treatment (Engstrom et al., 2003), and in parvocellular paraventricular nucleus neurones proenkephalin-A mRNA expression is rapidly induced by some stressors (Harbuz et al., 1991). How allopregnanolone might up-regulate opioid mechanisms in the NTS is not clear: interaction with GABAA receptors is a possibility. Such an interaction regulates oxytocin gene expression at the end of pregnancy (Blyth et al., 2000), and the expression of specific GABAA receptor subunit mRNAs (Concas et al., 1998; Follesa et al., 2004), including in the supraoptic nucleus (Fenelon and Herbison, 2000). Furthermore, GABA action through GABAA receptors regulates neuropeptide gene expression in the paraventricular nucleus (Bali and Kovacs, 2003).

␤-Endorphin and arcuate nucleus. Although there are indications of increased activity of arcuate nucleus ␤-endorphin (pro-opiomelanocortin mRNA-expressing) neurones in late pregnancy (Douglas et al., 2000), a role in suppressing oxytocin neurone responses to stressors in pregnancy is not established. Somato-dendritic oxytocin release and actions. Oxytocin released in the magnocellular nuclei has local positive feedback actions on oxytocin neurones during parturition and lactation (see Russell et al., 2003). Stressors increasing somato-dendritic oxytocin release include systemic cholecystokinin (Neumann et al., 1994) and forced swimming (Wigger and Neumann, 2002). Interleukin-1␤ (given centrally) stimulates oxytocin release in the supraoptic nucleus (Landgraf et al., 1995), but effects of systemic interleukin-1␤ are not known. Noradrenaline stimulates somato-dendritic oxytocin release (Lipschitz et al., 2004), but confirmation is needed that the failure of interleukin-1␤ to stimulate noradrenaline release in the magnocellular nuclei in late pregnancy (Brunton et al., 2005) is accompanied by failure of somato-dendritic oxytocin release, and that this is reversed by naloxone. Conversely, oxytocin increases noradrenaline release in the supraoptic nucleus (Onaka et al., 2003). Naloxone increases oxytocin release within the supraoptic nucleus in late pregnant, but not virgin, rats under basal conditions, indicating that opioids tonically inhibit somato-dendritic release of oxytocin in late pregnancy (Douglas et al., 1995). It also remains to be shown whether in late pregnancy finasteride restores interleukin-1␤ responses with respect to noradrenaline release, oxytocin neurone firing-rate and possibly somato-dendritic oxytocin release, as well as oxytocin secretion (Fig. 2a, b). Surprisingly, brief exposure of rat supraoptic neurones to a supraphysiological allopreg-

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-GABA

Glia

(+)EOP

-AP P

NA+

Dendrite

VIRGIN

-GABA

P

Dendrite

OXYTOCIN neurone PREGNANT: term -1 to 2 days

IL-1β β

Blood vessels

PENK-A/µ µ-OR↑

NA+

- EOP↑

-AP

AP?

X-2 CO

β-END Arcuate P nucleus Axon terminals - κ-opioid Posterior pituitary

OXYTOCIN neurone

Glia

NTS (A2)

+ PG

NTS (A2)

- β-END

+ PG AP?

X-2 CO

IL-1β β Blood vessels

Arcuate P nucleus Axon terminals κ-opioid ↓ Posterior pituitary

Fig. 4. Summary of interleukin-1␤ pathway to oxytocin neurones: opioid and allopregnanolone (AP) actions in pregnancy. (a) In virgin rats: circulating interleukin-1␤ (IL-1␤) acts via IL-1 receptors on endothelial cells of blood vessels (star), activating cyclo-oxygenase 2 (COX-2) and prostaglandin (PG) synthesis. Via EP4 receptors, PG stimulates noradrenergic (NA; A2) neurones in NTS projecting to magnocellular oxytocin neurones in the paraventricular and supraoptic nuclei. AP, a progesterone (P) metabolite, can suppress oxytocin neurone responses to IL-1␤, but little is produced. Endogenous opioids (EOP) weakly facilitate centrally, but inhibit oxytocin release at the posterior pituitary. (b) In late pregnancy: NTS neurones are activated by i.v. IL-1␤, but noradrenaline (NA) is not released, so oxytocin neurones are not excited. Preterminal inhibition of NA release, probably by opioid produced by A2 neurones (pro-enkephalin-A (PENKA) and ␮-opioid receptor (␮-OR) mRNA expression are up-regulated in the NTS), prevents oxytocin neurone activation; ␤-endorphin (␤-END) might be involved. Posterior pituitary opioid mechanisms are down-regulated. Endogenous AP inhibits oxytocin responses to i.v. IL-1␤, via GABAA receptors on oxytocin neurones, and, or, by inducing expression of opioid mechanisms in NTS neurones.

nanolone concentration (100 nM) in vitro slightly increases somato-dendritic oxytocin release, but this action is not via GABAA receptors (Widmer et al., 2003). However, blocking allopregnanolone action on GABAA receptors with finasteride in late pregnancy may contribute to the oxytocin secretory response to interleukin-1␤ by simulating inhibitory effects of increased somato-dendritic release of oxytocin on GABA transmission (Hirasawa et al., 2004; Koksma et al., 2003). This would liberate a local positive feedback circuit.

CONCLUSION In late pregnancy, oxytocin neurone responses to systemic interleukin-1␤ are suppressed by allopregnanolone and endogenous opioids. Allopregnanolone may act independently, via GABAA receptors on oxytocin neurones, and by inducing opioid expression acting on the noradrenergic input from the medulla (Fig. 4). Abrogation of these mechanisms might predispose to stimulation of oxytocin secretion by infection, through cytokines, and hence pre-term labor. The mechanisms of reduced responses of oxytocin neurones to interleukin-1␤ in late pregnancy proposed here also underlie reduced HPA axis responses to inter-

leukin-1␤ (Brunton et al., 2004a, 2005), and might also explain reduced febrile responses in late pregnancy (Mouihate et al., 2002). Acknowledgments—Supported by the BBSRC. Prof. J. I. Mason for expert advice. Colin Weekes assisted with some experiments. Helen Cameron gave skilled technical assistance.

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(Accepted 8 September 2005) (Available online 28 November 2005)