Central Opioid Inhibition of Neuroendocrine Stress Responses in Pregnancy in the Rat Is Induced by the Neurosteroid Allopregnanolone

The hypothalamus–pituitary–adrenal (HPA) axis is the major neuroendocrine stress response system. Corticotropin-releasing hormone (CRH) neurons in the parvocellular paraventricular nucleus (pPVN) play a key role in coordinating responses of this system to stressors. The cytokine interleukin-1β (IL-1β), mimicking infection, robustly activates these CRH neurons via a noradrenergic input arising from the nucleus tractus solitarii (NTS). In late pregnancy, HPA axis responses to stressors, including IL-1β, are attenuated by a central opioid mechanism that auto-inhibits noradrenaline release in the PVN. Here we show that the neuroactive progesterone metabolite allopregnanolone induces these changes in HPA responsiveness to IL-1β in pregnancy. In late pregnancy, inhibition of 5α-reductase (an allopregnanolone-synthesizing enzyme) with finasteride restored HPA axis responses (rapidly increased pPVN CRH mRNA expression, ACTH, and corticosterone secretion) to IL-1β. Conversely, allopregnanolone reduced HPA responses in virgin rats. In late pregnancy, activity of the allopregnanolone-synthesizing enzymes (5α-reductase and 3α-hydroxysteroid dehydrogenase) was increased in the hypothalamus as was mRNA expression in the NTS and PVN. Naloxone, an opioid antagonist, restores HPA axis responses to IL-1β in pregnancy but had no additional effect after finasteride, indicating a causal connection between allopregnanolone and the endogenous opioid mechanism. Indeed, allopregnanolone induced opioid inhibition over HPA responses to IL-1β in virgin rats. Furthermore, in virgin rats, allopregnanolone treatment increased, whereas in pregnant rats finasteride decreased proenkephalin-A mRNA expression in the NTS. Thus, in pregnancy, allopregnanolone induces opioid inhibition over HPA axis responses to immune challenge. This novel opioid-mediated mechanism of allopregnanolone action may alter regulation of other brain systems in pregnancy.

In late pregnancy, reduced activation of pPVN CRH neurons by stressors leads to attenuated ACTH and corticosterone responses . This adaptation may be important in altered metabolic and immune balance in pregnancy and in protecting fetuses from adverse programming . In late pregnant rats, HPA axis responses to systemic IL-1␤ are essentially absent, through inhibition by an emergent endogenous opioid mechanism (Brunton et al., 2005). Systemic IL-1␤ still activates NTS neurons in pregnancy, but opioid acting presynaptically blocks IL-1␤-induced noradrenaline release in the PVN (Brunton et al., 2005). The source of the opioid peptide and receptor is evidently the NTS neurons that project to the PVN: mRNA expression for proenkephalin-A (pENK-A) and the -opioid receptor (MOR) is increased in the NTS A2 region in late pregnancy (Brunton et al., 2005).
Here we sought to identify the pregnancy signal that induces opioid inhibition of the HPA axis. The female sex steroids estradiol and progesterone are produced in increasing amounts in pregnancy, but mimicking their pregnancy levels in virgin rats does not attenuate HPA axis stress responses (Douglas et al., 2000). However, allopregnanolone (3␣,5␣tetrahydroprogesterone or 5␣-pregnan-3␣-ol-20-one) (AP), the neuroactive steroid metabolite of progesterone, is abundant in the brain in pregnancy and can reduce stress-induced HPA axis activity in male rats (Concas et al., 1998). Allopregnanolone is produced by sequential action of 5␣-reductase and 3␣-hydroxysteroid dehydrogenase (3␣-HSD), which are both expressed in the brain (Compagnone and Mellon, 2000). As well as allosterically modulating GABA A receptors, to enhance the effectiveness of GABA actions (Herd et al., 2007), allopregnanolone can also modulate neuropeptide gene expression (Bali and Kovács, 2003).
Here, we investigate a role for allopregnanolone in restraining HPA axis responses to IL-1␤ in late pregnancy by inhibiting allopregnanolone synthesis in late pregnant rats and by measuring HPA responses to IL-1␤ in nonpregnant rats treated with allopregnanolone. Furthermore, we test whether activity and gene expression of the allopregnanolone synthesizing enzymes are increased centrally in late pregnancy. Moreover, we seek evidence that allopregnanolone is involved in the induction and maintenance of the inhibitory opioid mechanism that emerges in pregnancy.

Materials and Methods
Animals. Female Sprague Dawley rats (initial body weight, 240 -290 g; Charles River Laboratories) were housed under standard conditions of temperature (20 -22°C) and lighting (12 h light/dark cycle, lights on at 7:00 A.M.) with food and water available ad libitum. Rats were initially housed in groups of five to six for 3 weeks and then caged individually after surgery. Pregnant rats were obtained by mating overnight with a sexually experienced male, and the pregnancy was confirmed by finding a vaginal plug of semen on the floor of the breeding cage the following morning (day 1 of pregnancy; day 22 is the expected day of parturition). Virgin rats selected at random stages of the estrus cycle were used as controls. The number of rats per group for each experiment is given in the figure legends. All procedures were performed in accordance with current United Kingdom Home Office regulations and with University of Edinburgh Ethical Committee approval.
Surgery: jugular vein cannulation. Changes in ACTH and corticosterone secretion were assessed by collecting serial venous blood samples. Five days before the experiment, virgin and day 16 pregnant rats were fitted with a SILASTIC jugular vein cannula (wall, 0.25 mm; internal diameter, 0.5 mm; filled with sterile heparinized 0.9% saline, 50 U/ml) under inhalational halothane anesthesia (2-3% halothane, in 1200 ml/ min oxygen) for subsequent blood sampling and drug administration.
Blood sampling and tissue collection. On the morning of the experiment (between 7:30and 9:00 A.M.), the jugular cannula was connected to polyvinyl chloride extension tubing (wall, 1 mm; internal diameter, 0.5 mm) filled with heparinized saline (50 U/ml 0.9% saline) and attached to a 1 ml syringe, for remote sampling. Rats were then left undisturbed for 90 min. After collection of two basal blood samples (at t ϭ Ϫ31 and Ϫ1 min), rats were administered 500 ng/kg human recombinant IL-1␤ intravenously (1 g/ml; dissolved in 0.2% bovine serum albumin in PBS; t ϭ 0 min; R & D Systems). Additional blood samples were taken 15, 30, 60, 90, and 120 min after treatment. Blood samples (0.35 ml) were collected into 1 ml syringes containing 20 l of 5% EDTA, chilled on ice, and centrifuged to separate plasma, which was frozen and stored at Ϫ20°C until assay. Each blood sample was replaced immediately with an equiv-alent volume of warmed sterile 0.9% saline. Rats were killed by conscious decapitation 4 h after IL-1␤ injection [the optimum time point for detecting peak changes in CRH mRNA expression after exposure to stress (Harbuz and Lightman, 1989)]. Trunk blood was collected into tubes containing chilled 5% EDTA (0.2 ml/100 g body weight), and plasma was separated and stored as above. Brains were rapidly removed, frozen on dry ice, and stored at Ϫ70°C until sectioning. CRH neuron activation was evaluated by quantitative in situ hybridization (ISH) for CRH mRNA (see below). Pregnancy status was checked postmortem, and only pregnant rats with at least six viable fetuses were included in the study (actual range, 6 -17 pups; mode, 14 pups).
Experiment 1: is allopregnanolone involved in suppressing HPA axis responses in pregnant rats? To investigate a role for allopregnanolone in suppressed HPA axis responses to IL-1␤ in late pregnancy, allopregnanolone production was blocked using finasteride (5␣-reductase inhibitor). Blood samples and brains were collected (as above) from virgin and pregnant (day 21) rats treated with either finasteride (25 mg/kg, s.c.; Steraloids) or vehicle (15% ethanol in sesame oil; 0.5 ml/kg) 20 and 2 h before IL-1␤ (t ϭ Ϫ20 and Ϫ2 h). A separate group of pregnant (day 21) rats were treated with finasteride or vehicle (as above) only (no IL-1␤). Such finasteride treatment reduces brain allopregnanolone content by 90% in pregnant and 77% in nonpregnant rats, to similar actual levels (Concas et al., 1998).
Experiment 2: can the effects of pregnancy on HPA axis responses be mimicked in virgin rats with allopregnanolone treatment? To test whether suppressed HPA axis responses to IL-1␤ could be mimicked in virgin rats with allopregnanolone treatment, blood samples and brains were collected from virgin rats treated with AP (3 and 1 mg/kg, s.c., 20 and 2 h before IL-1␤, respectively; Steraloids) or vehicle (15% ethanol in corn oil; 0.5 ml/kg). To verify that the effects of finasteride on HPA responses to IL-1␤ seen in pregnant rats were attributable to the actions of allopregnanolone, blood samples and brains were also collected from pregnant (day 20 -21) rats treated with finasteride (as before) and either allopregnanolone or vehicle (as above).
Experiment 3: can the allopregnanolone precursors suppress HPA axis responses in virgin rats? To establish whether administration of the allopregnanolone precursors progesterone or dihydroprogesterone (DHP) can restrain HPA axis responses to IL-1␤, virgin rats were treated with progesterone (20 and 4 mg/kg, s.c., 20 and 2 h before IL-1␤, respectively; Steraloids) or vehicle (10% benzyl alcohol and 0.3% cresol in arachis oil; 0.5 ml/kg). A separate set of rats were treated with dihydroprogesterone (4 and 1 mg/kg, s.c. 20 and 2 h before IL-1␤, respectively; Steraloids) or vehicle (15% benzyl alcohol and 0.3% cresol in arachis oil; 0.5 ml/kg). In both cases, blood and brains were collected to evaluate HPA axis activity.
Experiment 4: does the expression of mRNAs for the allopregnanolonesynthesizing enzymes increase in the brain during pregnancy? To investigate whether mRNA expression for 5␣-reductase and/or 3␣-HSD is increased in the brain in pregnancy, untreated virgin and pregnant (day 21) rats were killed by conscious decapitation. Brains, brainstems, and liver (positive control) were removed, frozen on dry ice, and stored at Ϫ70°C until processing for ISH.
Experiment 5: are the activities of the allopregnanolone-synthesizing enzymes increased in the brain during pregnancy? To investigate whether the activity of 5␣-reductase and/or 3␣-HSD is increased in the brain in pregnancy, untreated virgin and pregnant (day 21) rats were killed by conscious decapitation. Brains were removed, and the hypothalamus (cut rostrocaudally between the optic chiasm and mammillary bodies, laterally at the hypothalamic sulci and dorsally below the ventral thalamus) and medulla oblongata [bregma, Ϫ10 to Ϫ15 mm (Paxinos and Watson, 1998)] were dissected, frozen on dry ice, and stored at Ϫ70°C until subsequent enzyme assay.
Experiment 6: are the effects of allopregnanolone and endogenous opioids in pregnant rats interdependent? Pregnant rats (day 21) were pretreated with either vehicle or finasteride (as before). Immediately after the first basal blood sample was collected, rats were given either naloxone [5 mg/kg, i.v.; naloxone was used at this dose as a nonselective opioid receptor antagonist, as before (Brunton et al., 2005), because the opioid receptor type that mediates opioid inhibition of HPA axis in pregnancy has not been defined] or vehicle (0.9% saline, 0.5 ml/kg). After an addi-tional two blood samples 15 and 30 min later, all rats were given IL-1␤ (as before). Additional blood samples were taken 15, 30, 60, 90, and 120 min after IL-1␤, rats were killed at 4 h (as before), and brains were collected to assess CRH neuron activation by quantitative ISH for CRH mRNA. One group of control virgin rats were given oil vehicle and saline pretreatment before IL-1␤ and sampled as above. Separate groups of virgin and pregnant rats were blood sampled after treatment with only naloxone (i.e., with no subsequent IL-1␤ treatment) or saline.
Experiment 7: can allopregnanolone induce opioid inhibition over HPA axis responses in virgin rats? To establish whether allopregnanolone exerts its effects on HPA responsiveness by inducing inhibitory opioid tone, virgin rats were given either allopregnanolone or vehicle (as before). Immediately after the first basal blood sample was taken, rats were given either naloxone (5 mg/kg, i.v.) or vehicle (0.9% saline, 0.5 ml/kg). After an additional two blood samples 15 and 30 min later, all rats were given IL-1␤ (as before). Additional blood samples were taken 15, 30, 60, 90, and 120 min after IL-1␤, rats were killed at 4 h (as before), and brains were collected to assess CRH neuron activation by quantitative ISH for CRH mRNA.
Experiment 8: does allopregnanolone alter central opioid expression? To investigate whether allopregnanolone upregulates central opioid expression virgin rats were treated with allopregnanolone or vehicle (as in experiment 2). Late pregnant rats were treated with finasteride or vehicle (as in experiment 1). Two hours after the second injection, rats were killed by conscious decapitation. Expression of pENK-A, proopiomelanocortin (POMC), and MOR mRNAs in selected nuclei in the hypothalamus and brainstem were evaluated by quantitative ISH.
Radioimmunoassays. ACTH concentrations were determined in unextracted plasma using a commercially available two-site immunoradiometric kit (Euro-diagnostica), whereas corticosterone (IDS Ltd.) and progesterone (Diagnostic Systems Laboratories) were measured using radioimmunoassay kits. The sensitivity and intra-assay variation for each of the assays were as follows: ACTH: sensitivity, 1 pg/ml and intra-assay variation, Ͻ7%; corticosterone: sensitivity, 0.4 ng/ml and intra-assay variation Ͻ8%; progesterone: sensitivity, 0.3 ng/ml and intra-assay variation Ͻ6%. All samples from any one experiment were assayed together.
In situ hybridization. Tissue was sectioned coronally on a cryostat at 15 m and thaw mounted onto Polysine slides. All sections from a particular experiment were processed in the same hybridization reaction. 35 S-Radiolabeled oligonucleotide probes (MWG-Biotech) were used to detect CRH, pENK-A, POMC, and -opioid receptor mRNA expression. The sequences of each oligonucleotide probe are given in Table 1 and in situ hybridization using these probes was performed as described previously (Brunton et al., 2005).
[ 35 S]UTP-labeled cRNA sense and antisense probes were synthesized from the linearized pBluescript SKϪ vector expressing a 344 bp (nucleotides 991-1334) cDNA fragment encoding rat 5␣-reductase type I [the predominant isoform expressed in adult brain (Compagnone and Mellon, 2000;Sánchez et al., 2008a,b); generously provided by Prof. Marcel Karperien, Leiden University Medical Center, Leiden, The Netherlands]. The plasmid was linearized with XhoI and XbaI and transcribed using T3 and T7 polymerase (Promega) for the sense and antisense riboprobes, respectively. To detect 3␣-HSD mRNA, [ 35 S]UTP-labeled sense and antisense riboprobes were generated from the linearized pGEM3 vector expressing a 982 bp (nucleotides Ϫ129 to ϩ853) cDNA fragment encoding rat 3␣-HSD (generously provided by Prof. Trevor Penning, University of Pennsylvania, Philadelphia, PA). The plasmid was linearized with SalI and SspI, and sense and antisense cRNAs incorporating [ 35 S]UTP were transcribed from the T7 and SP6 promoters, respectively.
For in situ hybridization using riboprobes, tissue was first fixed in 4% paraformaldehyde, acetylated in triethanolamine/acetic anhydride solution, and then partially dehydrated in ethanol before incubation in prehybridization buffer [50% deionized formamide (v/v), 1ϫ Denhardt's solution, 1 mM EDTA, 0.5 mg/ml salmon testes DNA, 600 mM sodium chloride, 10 mM Tris, and 125 g/ml yeast tRNA] at 50°C for 120 min. Riboprobes incorporating 35 S-labeled UTP were mixed with hybridization buffer [50% deionized formamide (v/v), 1ϫ Denhardt's solution, 10% dextran sulfate (w/v), 15 mM dithiothreitol, 1 mM EDTA, 0.1 mg/ml salmon testes DNA, 600 mM sodium chloride, 10 mM Tris, and 125 g/ml yeast tRNA], heated for 10 min at 70°C, quenched on ice, and applied to the tissue (650,000 cpm/ section). To aid tissue penetration, the radiolabeled 3␣-HSD riboprobe was fragmented by alkaline hydrolysis and neutralized before hybridization. Sections were hybridized at 55°C and 60°C (for 5␣-reductase and 3␣-HSD mRNAs, respectively) for 18 -19 h. Sections of rat liver treated as above were used as positive controls. Brain and liver sections serving as negative controls to ensure probe specificity were hybridized with [ 35 S]UTP-labeled cRNA sense probes. The hybridization signal over tissue hybridized with the sense probe was not different from background.
After hybridization, the slides were rinsed briefly (to remove coverslips) in 2ϫ SSC and then washed three times for 5 min in 2ϫ SSC at room temperature. Next, sections were incubated in buffer (1 mM EDTA, 500 mM sodium chloride, and 10 mM Tris) containing 15 g/ml RNase A for 60 min at 37°C. Sections were then briefly rinsed in 2ϫ SSC at room temperature before additional stringency washing (for 5␣-reductase, once in 2ϫ SSC at 37°C and twice at 55°C for 30 min each, followed by two 60 min washes in 0.2ϫ SSC at 55°C; and for 3␣-HSD, 60 min each in 2ϫ SSC at 50°C and in 0.2ϫ SSC at 55°C and then 60°C). After the posthybridization washes, tissue was dehydrated in an ascending series of ethanol containing 300 mM ammonium acetate, air dried, dipped in autoradiographic emulsion (Ilford K-5), and exposed at 4°C. Exposure times for each mRNA species were as follows: CRH, 10 weeks; 3␣-HSD, 10 weeks; pENK-A, 6 weeks; POMC, 5 weeks; -opioid receptor, 6 weeks; and 5␣-reductase, 7 weeks.
Slides were developed (Kodak D-19; Sigma), fixed (Hypam rapid fixer; Ilford), and counterstained with hematoxylin and eosin. Autoradiographs were quantified using three methods. (1) The area of each region of interest (e.g., PVN or NTS) and the silver grain area overlying the region were measured (NIH Image software, version 1.62) and are presented as grain area/brain area (mm 2 /mm 2 ). Background measurements were made over areas adjacent to the region of interest and subtracted.
(2) The number of cells positively hybridized in a particular brain region was counted manually in emulsion-dipped sections. A positive cell was defined as one with more overlying silver grains than the mean over 10 cells lateral to the region of interest (background) ϩ 3 SDs. Data are presented as the number of positive cells per brain area (mm 2 ). (3) Grain density per cell data were determined by measuring the silver grain area over ϳ240 cells per area per rat (Openlab version 2 software; Improvision). Background measurements were made over equivalently sized cells in the neuropil adjacent to the region of interest and subtracted. In each case, measurements were made over a minimum of 12 PVN or NTS profiles (bilateral measurements in six consecutive sections). Integrated density was calculated by multiplying the mean number of positive neurons in a specific brain region section by the mean grain area per cell (data given in arbitrary units). For all ISH measurements, average values for each rat were used to calculate group means Ϯ SEM.
Enzyme assays and thin-layer chromatography. Brain tissue was weighed, and a 10% (w/v) homogenate (ϳ12.5 mg protein/ml) was prepared in buffer, pH 7.22, containing 100 mM potassium phosphate, 1 mM EDTA, and 0.32 M sucrose using a glass homogenizer. Protein concentration of the homogenates was measured using the Bradford protein assay. Homogenates were preincubated at 37°C for 10 min in 100 mM potassium phosphate buffer containing 50 M EDTA, 5 mM dithiothreitol, and the appropriate cofactor (100 M NADPH for 5␣-reductase and 100 M NADPH and 100 M NADH for 3␣-HSD) before addition of 20 Ci [1,2,6,7-3 H(N)]-progesterone (specific activity, 115Ci/mmol). The preparations were incubated in a shaking water bath for 40 min at 37°C. Control reactions were included for each assay and contained all of the components (given above) except for the tissue homogenate. The reaction was terminated by transferring the tubes to ice and by the addition of ice-cold methanol/chloroform (1:1). Tubes were agitated for 5 min, and the different phases were separated by centrifugation (6000 rpm for 1 min). Progesterone metabolites were separated by thin-layer chromatography (TLC) using silica gel 60 F 254 TLC plates (20 ϫ 20 cm; Merck) previously activated by heating at 100°C for 15 min. Fifty microliters of each extract or unlabeled standard [1 mg/ml (w/v) progesterone, dihydroprogesterone, and allopregnanolone in ethanol] were spotted onto the bottom right-hand corner of the TLC plates. Each plate was run in a two-dimensional system: twice in solvent system 1 (benzene/methanol, 19:1, v/v), then plates were turned 90 o and run twice in solvent system 2 (cyclohexane/ ethylacetate, 1:2, v/v). Steroids were visualized by first exposing the TLC plates to 30% H 2 SO 4 in ethanol, heating to 140°C for 15 min, and then by ultraviolet illumination. The spots corresponding to the substrate (progesterone) and metabolites were scraped from the plates and the radioactivity counted in a ␤-scintillation counter. The quantity of the metabolites converted from progesterone is expressed as a percentage of the total radioactivity and is indicative of the enzyme activities. Statistical analysis. SigmaStat (version 3.1; Systat Software) and SPSS software (version 17; SPSS) were used to analyze the data. The specific statistical tests used are given in Results. Data from in situ hybridization studies and enzyme activity data were analyzed by ANOVA or Student's t test; when we had predicted direction of change, a one-tailed Student's t test was used. To analyze the effects of treatment across time in plasma hormone data, two-way repeated measures ANOVA (two-way RM ANOVA) (SigmaStat) were used when there were two treatment groups. In cases in which three or more treatment groups were to be analyzed, a repeated measures mixed-model ANOVA (mixed ANOVA) (SPSS) was used, followed by a two-way RM ANOVA to critically test our original hypotheses with planned comparisons when appropriate. Specific differences were isolated using Student-Newman-Keuls (SNK) multiple comparison post hoc tests. In each case, values shown are group means Ϯ SEM. p values Ͻ0.05 were considered statistically significant.
There was a significant effect of pregnancy (F (1,20) ϭ 6.0, p ϭ 0.023, mixed ANOVA) on plasma corticosterone concentrations (Fig. 1b). Finasteride treatment had no significant effect on plasma corticosterone concentration within the virgin groups (F (1,44) ϭ 0.176, p ϭ 0.683, two-way RM ANOVA), but there was a significant effect of finasteride treatment within the pregnant groups (F (1,42) ϭ 4.922, p ϭ 0.048). Basal plasma corticosterone concentration was greater in the pregnant rats (this only reached statistical significance in the preg/veh group; p ϭ 0.02, SNK test), but finasteride treatment had no effect in either virgin or pregnant rats (basal plasma corticosterone concentrations: virgin/ veh, 50.1 Ϯ 5.3 ng/ml; virgin/FIN, 54.3 Ϯ 6.5 ng/ml; preg/veh, 159.9 Ϯ 27.6 ng/ml; preg/FIN, 120.4 Ϯ 17.5 ng/ml). As expected, IL-1␤ significantly increased plasma corticosterone concentra- tions in the vehicle-treated virgin but not pregnant rats (Fig. 1b) ( p Ͻ 0.001, SNK test). Finasteride pretreatment had no significant effect on the corticosterone response to IL-1␤ in the virgin rats, but it resulted in a significant corticosterone response in the late pregnant rats (Fig. 1b). Treatment with finasteride alone (i.e., no IL-1␤) had no significant effect on basal CRH mRNA expression in the pPVN of late pregnant rats (grain area per pPVN was 0.055 Ϯ 0.013 mm 2 /mm 2 in vehicletreated rats, n ϭ 6 vs 0.050 Ϯ 0.019 mm 2 / mm 2 in finasteride-treated rats, n ϭ 4; p Ͼ 0.05, two-tailed Student's t test). There was, however, a significant effect of both pregnancy (F (1,18) ϭ 34.5, p Ͻ 0.001, twoway ANOVA) and finasteride treatment (F (1,18) ϭ 7.3, p ϭ 0.015) on CRH mRNA expression in the pPVN after IL-1␤ administration. In vehicle-treated pregnant rats, CRH mRNA expression in the pPVN was significantly less 4 h after systemic IL-1␤ than in the vehicle-treated virgin group (Fig. 1c,d). Finasteride had no effect on CRH mRNA expression after IL-1␤ in the virgin rats, but it resulted in a significant increase in expression in the late pregnant rats (Fig. 1c,d).

Effect of allopregnanolone treatment on HPA axis responses to IL-1␤
There was a significant effect of allopregnanolone treatment (F (1,66) ϭ 13.2, p ϭ 0.003, two-way RM ANOVA) on ACTH secretion in the virgin rats (Fig. 2a). There was no difference in basal plasma ACTH concentration between vehicle and allopregnanolone-pretreated virgin rats (Fig. 2a). IL-1␤ significantly increased ACTH secretion within 15 min in both the vehicle-treated virgins (4.0-fold increase; p Ͻ 0.001, SNK test) and the allopregnanolone treated virgins (2.0-fold increase; p Ͻ 0.001); however, the response was markedly reduced in the allopregnanolone-treated group (57% of the peak response in the vehicle group; p Ͻ 0.001).
There was also a significant effect of allopregnanolone treatment on corticosterone secretion in the virgin rats (F (1,51) ϭ 5.1, p ϭ 0.028, two-way RM ANOVA) (Fig. 2c). Consistent with the ACTH data, basal corticosterone secretion was not significantly different between the vehicleand allopregnanolone-treated virgins (Fig.  2c), but the IL-1␤-evoked increase in corticosterone secretion was significantly attenuated in the allopregnanolone-treated group (Fig. 2c).
In the late pregnant rats, there was a significant effect of drug treatment (F (1,58) ϭ 6.2, p ϭ 0.034, two-way RM ANOVA) on plasma ACTH concentration (Fig. 2b). Neither finasteride alone nor combined finasteride and allopregnanolone treatment affected basal plasma ACTH concentration in late pregnant rats (Fig. 2b). As before, blocking allopregnanolone production with finasteride restored a significant ACTH response to IL-1␤ in late pregnant rats (Fig. 2b). This  rats (a, c, e) were treated with allopregnanolone (3 and 1 mg/kg, s.c., 20 and 2 h before IL-1␤, respectively) or vehicle (veh), whereas pregnant (day 20 -21) rats (b, d, f) were treated with finasteride (25 mg/kg, s.c.) and either allopregnanolone (as above) or vehicle 20 and 2 h before IL-1␤. After two basal (B) blood samples (at t ϭϪ31 and Ϫ1 min), all rats were administered IL-1␤ (500 ng/kg, i.v.) at t ϭ 0 min. Additional blood samples were taken 15, 30, 60, 90, and 120 min after treatment. Trunk blood was collected at t ϭ 240 min. Plasma ACTH concentrations in virgin (a) and pregnant (b) rats. c, d, Plasma corticosterone concentrations in virgin (c) and pregnant (d) rats. n ϭ 5-7 rats per group. a, *p Ͻ 0.03 versus basal values in the same group; # p Ͻ 0.04 versus virgin/veh group; b, *p Ͻ 0.05 versus basal values in the same group; # p Ͻ 0.001 versus preg/FIN/AP group; c, d, *p Ͻ 0.001 versus basal values in the same group; # p Ͻ 0.04 versus other group at the same time point. Rats were killed 4 h after IL-1␤ administration, and brains were processed by in situ hybridization. e, f, Quantification of CRH mRNA expression in the pPVN in brains from virgin (e) and pregnant (f) rats. Grain density per pPVN profile [expressed as arbitrary units (a.u.)] was calculated by multiplying the mean grain density per neuron by the mean number of neurons positively expressing CRH mRNA. n ϭ 6 -9 rats per group. e, *p Ͻ 0.001 versus virgin/AP group; f, *p Ͻ 0.02 versus preg/FIN/AP group. All values are group means Ϯ SEM. effect was prevented in the pregnant rats by coadministration of allopregnanolone.
There was a significant effect of drug treatment (F (1,40) ϭ 20.6, p ϭ 0.002, twoway RM ANOVA) on corticosterone secretion in the pregnant rats. Accordingly, finasteride treatment restored a corticosterone response to IL-1␤ in late pregnant rats (Fig. 2d), which was attenuated by combined allopregnanolone treatment.
CRH mRNA expression in the pPVN 4 h after systemic IL-1␤ was significantly less in virgin rats pretreated with allopregnanolone compared with those pretreated with vehicle ( Fig. 2e) ( p Ͻ 0.001, one-tailed Student's t test). Furthermore, CRH mRNA expression in the pPVN 4 h after systemic IL-1␤ was significantly less in pregnant rats treated with a combination of finasteride and allopregnanolone compared with pregnant rats treated with finasteride alone (Fig. 2f ) ( p ϭ 0.01). Treatment of virgin rats with allopregnanolone only for 20 h had no effect on basal CRH mRNA expression in the pPVN ( Table 2).

Figure 3.
Effect of progesterone or dihydroprogesterone treatment on HPA axis responses to IL-1␤ in virgin rats. a, b, Virgin rats were treated with either progesterone (prog; 20 and 4 mg/kg, s.c., 20 and 2 h before IL-1␤, respectively) or vehicle 20 and 2 h before IL-1␤. c, d, In a separate experiment, rats were treated with DHP (4 and 1 mg/kg, s.c., 20 and 2 h before IL-1␤, respectively) or vehicle (veh) 20 and 2 h before IL-1␤. After two basal blood samples (B; at t ϭ Ϫ31 and Ϫ1 min), all rats were administered IL-1␤ (500 ng/kg, i.v.), and additional blood samples were withdrawn 15, 30, 60, 90, and 120 min after treatment. n ϭ 5-8 rats per group. Values are group means Ϯ SEM. Plasma ACTH concentration (a) and plasma corticosterone concentration (b) before and after systemic IL-1␤ in vehicle-and progesterone-treated virgin rats. Plasma ACTH concentration (c) and plasma corticosterone concentration (d) before and after systemic IL-1␤ in vehicle-and DHP-treated virgin rats. *p Ͻ 0.05 versus basal.
pression in the PVN between virgin and late pregnant rats (Fig.  4a), but 5␣-reductase mRNA levels were significantly greater in the A2 region of the NTS of the late pregnant group compared with the nonpregnant group (Fig. 4b,f ) ( p ϭ 0.047, one-tailed Student's t test). There was a modest but significant increase in 3␣-HSD mRNA expression in the PVN in late pregnant rats compared with virgin rats (Fig. 4c,e) ( p ϭ 0.042), but a significant difference in the level of 3␣-HSD mRNA expression in the A2 region of the NTS was not detected between virgin and pregnant rats (Fig. 4d) ( p ϭ 0.069).
Activity of the progesterone converting enzymes in the brain in late pregnancy 5␣-Reductase activity was significantly greater in the hypothalamus in late pregnant rats compared with virgin rats ([ 3 H]progesterone conversion into DHP was 2.6 Ϯ 0.5% in the virgin group, n ϭ 8 vs 5.6 Ϯ 0.8% in the late pregnant group, n ϭ 6; p ϭ 0.033, one-tailed Student's t test). 3␣-HSD activity also tended to be greater in the hypothalamus in pregnant rats, but this was not statistically significant ([ 3 H]progesterone conversion into AP was 0.3 Ϯ 0.1% in the virgin group, n ϭ 8 vs 0.7 Ϯ 0.3% in the late pregnant group, n ϭ 6; p ϭ 0.07). No difference was detected in the activities of either enzyme in the medulla oblongata ([ 3 H]progesterone conversion into DHP was 2.6 Ϯ 0.7% in the virgin group vs 3.2 Ϯ 0.8% in the late pregnant group, p Ͼ 0.05; [ 3 H]progesterone conversion into AP was 0.3 Ϯ 0.1% in the virgin group vs 0.4 Ϯ 0.1% in the late pregnant group, p Ͼ 0.05).

Effect of blocking allopregnanolone synthesis and endogenous opioid action on HPA axis responses to IL-1␤ in late pregnant rats
There was a significant effect of finasteride (F (1,20) ϭ 13.9, p Ͻ 0.001, mixed ANOVA) and naloxone (F (1,20) ϭ 4.4, p ϭ 0.048) treatment on ACTH responses to IL-1␤ in the late pregnant groups (Fig. 5a). Neither finasteride nor naloxone altered basal plasma ACTH concentration in virgin or pregnant rats (Fig. 5a). IL-1␤ significantly increased plasma ACTH concentration (3.2fold increase) (Fig. 5a,b) in the vehicle-pretreated virgin rats but had no significant effect in the vehicle-pretreated pregnant rats (Fig. 5a,b). Both finasteride only and naloxone only pretreatment restored an ACTH response to IL-1␤ in the late pregnant rats to 71 and 65% of the response observed in the virgin controls, respectively ( Fig. 5a,b). However, combined finasteride and naloxone pretreatment had no additional effect on the ACTH response to IL-1␤ in late pregnant rats compared with either naloxone or finasteride treatment alone (Fig. 5a,b). In a separate experiment, naloxone (5 mg/kg, i.v., with no subsequent IL-1␤ treatment) had no effect on plasma ACTH concentration over a 90 min period in either virgin or late pregnant rats (Fig. 5c) ( p Ͼ 0.05, mixed ANOVA). Plasma corticosterone concentrations before IL-1␤ administration were as follows: virgin/oil/saline, 106.6 Ϯ 11.5 ng/ml; pregnant/oil/saline, 121.4 Ϯ 8.9 ng/ml; pregnant/oil/naloxone, 129.2 Ϯ 10.1 ng/ml; pregnant/finasteride/saline, 107.0 Ϯ 9.7 ng/ ml; pregnant/finasteride/naloxone, 117.4 Ϯ 8.9 ng/ml. There was an overall effect of drug treatment (F (4,158) ϭ 4.6, p ϭ 0.006, two-way RM ANOVA) on corticosterone responses to IL-1␤. IL-1␤ significantly increased plasma corticosterone concentration in the vehicle-treated virgin but not pregnant rats (Fig. 5d,e). Pretreatment with finasteride alone, naloxone alone, or combined finasteride and naloxone all restored corticosterone responses to IL-1␤ in the pregnant groups, with no significant differences between groups or compared with the vehicle-treated virgin rats (Fig. 5d,e).
There was a significant effect of pregnancy status (F (1,31) ϭ 25.9, p Ͻ 0.001, two-way ANOVA) and drug treatment (F (3,31) ϭ 5.0, p ϭ 0.006) on CRH mRNA expression. CRH mRNA expression in the pPVN 4 h after systemic IL-1␤ was significantly less in vehicle-pretreated pregnant rats than in vehicle-treated virgin rats (Fig. 5f ). Pretreatment with finasteride alone, naloxone alone, or combined finasteride and naloxone all resulted after IL-1␤ in similar levels of CRH mRNA expression in the pPVN of late pregnant rats that were significantly greater than in the vehicle-treated pregnant group (Fig. 5f ). Naloxone alone (i.e., no subsequent IL-1␤ treatment) had no effect after 4 h on basal CRH mRNA expression in the pPVN (same rats as in Fig. 5c); the mean Ϯ SEM number of CRH mRNA-expressing cells in the pPVN was as follows: virgin/saline, 228.1 Ϯ 13.4 (n ϭ 10); virgin/ naloxone, 221.2 Ϯ 12.2 (n ϭ 5); pregnant/saline, 176.5 Ϯ 16.4 (n ϭ 10); pregnant/naloxone, 176.6 Ϯ 18.3 (n ϭ 6). There was no significant effect of naloxone on CRH mRNA in the virgin or the pregnant rats ( p Ͼ 0.05, two-way ANOVA), but there was an effect of pregnancy (F (1,27) ϭ 8.3, p ϭ 0.008). As detailed above, finasteride alone had no effect on CRH mRNA expression in the pPVN of late pregnant rats.

Effect of allopregnanolone treatment on induction of inhibitory opioid tone over HPA axis responses to IL-1␤ in virgin rats
There was a significant effect of the allopregnanolone treatment (F (1,109) ϭ 6.2, p ϭ 0.023, two-way RM ANOVA) within the saline-treated rats on plasma ACTH concentrations (Fig. 6a). Moreover, within the allopregnanolone-treated rats, there was a significant effect of naloxone treatment (F (1,105) ϭ 4.0, p ϭ 0.048). There was no difference in basal ACTH secretion among vehicle-and allopregnanolone-pretreated virgin rats with or without naloxone treatment (Fig. 6a). In the vehicle (oil)pretreated groups, IL-1␤ significantly increased plasma ACTH concentration, with no effect of previous naloxone treatment (Fig. 6a,b). As before (Fig. 2), IL-1␤ induced a significant increase in ACTH secretion in the AP/saline-treated group (2.2-fold increase), but the amplitude of the response was attenuated compared with the virgin control group (3.8-fold increase) (Fig. 6b). Moreover, the suppressive effect of AP on the ACTH response to IL-1␤ was completely reversed in the presence of naloxone (Fig.  6a,b). Similarly, CRH mRNA expression in the pPVN 4 h after IL-1␤ administration was less in the virgin rats treated with AP alone compared with the controls (Fig. 6c) (F (1,36) ϭ 5.5, p ϭ 0.025, two-way ANOVA). This effect of AP was reversed in rats coadministered naloxone (Fig. 6c).

Effect of allopregnanolone treatment on central opioid gene expression in virgin rats
Mimicking pregnancy in virgin rats with allopregnanolone treatment significantly increased pENK-A mRNA expression in the A2 region of the NTS (Fig. 7a,b) ( p Ͻ 0.001, one-tailed Student's t test). This was a consequence of both an increase in the number of neurons expressing pENK-A mRNA and an increase in the Figure 5. Effect of inhibiting allopregnanolone synthesis and endogenous opioid action on HPA axis responses to IL-1␤ in late pregnancy. Day 21 pregnant (preg) rats were treated with either finasteride (FIN; 25 mg/kg, s.c.) or vehicle (oil) 20 and 2 h before IL-1␤. After a basal (B) blood sample (at t ϭ Ϫ31 min), rats were administered naloxone (NLX; 5 mg/kg, i.v.) or vehicle [saline (sal); 0.5 ml/kg, i.v.]. Two additional blood samples were collected at Ϫ16 and Ϫ1 min, after which all rats were administered IL-1␤ (500 ng/kg, i.v.) at t ϭ 0 min. Additional blood samples were taken 15, 30, 60, 90, and 120 min after treatment. Trunk blood was collected at t ϭ 240 min. A group of virgin rats pretreated with vehicle (oil) and then saline before IL-1␤ administration was included for comparison. a, b, Plasma ACTH concentration (a) and increase in ACTH secretion (b) from basal levels 30 min after IL-1␤. c, Plasma ACTH concentration in a separate group of virgin and day 21 pregnant (preg) rats before and after treatment only with naloxone (NLX; 5 mg/kg, i.v.) or saline (0.5 ml/kg, i.v.) administration. No significant differences within or between groups were detected. d, e, Plasma corticosterone concentration (d) and increase in corticosterone secretion (e) from basal levels 30 min after IL-1␤ from the same rats shown in a and b. n ϭ 6 -7 rats per group. a, *p Ͻ 0.04 versus basal values in the same group; # p Ͻ 0.04, ϩ p Ͻ 0.007 versus all other groups at the same time point; b, *p Ͻ 0.001 versus all other groups; d, *p Ͻ 0.009 versus basal values in the same group; # p Ͻ 0.008 versus all other groups at the same time point; ϩ p Ͻ 0.02 versus preg/FIN/NLX group. e, *p Ͻ 0.05 versus all other groups. Rats were killed 4 h after IL-1␤ administration, and brains were processed by in situ hybridization. f, Quantification of CRH mRNA expression in the pPVN. n ϭ 6 -8 rats per group. f, *p Ͻ 0.05, # p Ͻ 0.001 versus preg/oil/saline group. All values are group means Ϯ SEM. levels of expression per neuron (Table 2). Allopregnanolone treatment had no significant effect on pENK-A mRNA expression in the pPVN, magnocellular division of the PVN or the arcuate nucleus (Table 2). There were also no significant differences in POMC mRNA expression in the arcuate nucleus or -opioid receptor mRNA expression in the NTS (Table 2). In late pregnant rats, 20 h finasteride treatment resulted in a significant reduction in pENK-A mRNA expression in the A2 region of the NTS (Fig. 7c) ( p ϭ 0.003).

Discussion
Our results demonstrate that allopregnanolone restrains HPA axis responses to immune challenge in late pregnancy and implicate mediation of allopregnanolone actions by activation of endogenous opioid expression in the NTS.

Role of allopregnanolone
HPA responses to IL-1␤ were suppressed in late pregnancy as found previously (Brunton et al., 2005). Blocking allopregnanolone production with finasteride [previously shown to decrease cortical allopregnanolone content by 90% (Concas et al., 1998)] restored HPA axis responses to IL-1␤ in pregnant rats, reflected by increased CRH mRNA expression in the pPVN, and ACTH and corticosterone secretion, whereas concurrent allopregnanolone treatment prevented these finasteride effects. Furthermore, we mimicked reduced pregnancy-type HPA axis responses to IL-1␤ in nonpregnant rats with short-term (2-20 h) allopregnanolone treatment. Central effects were shown because, in pregnant rats finasteride enhanced and in virgin rats allopregnanolone treatment reduced the increase in pPVN CRH mRNA expression after systemic IL-1␤. These treatments did not affect basal pPVN CRH mRNA expression or basal plasma ACTH or corticosterone concentrations. Hence, the neurosteroid manipulations specifically altered HPA responses to the systemic IL-1␤ challenge.
Systemic IL-1␤ activates pPVN CRH neurons via brainstem noradrenergic A2 neurons (Rivest et al., 2000) but is ineffective in late pregnancy (Brunton et al., 2005). It is unclear where in the brain allopregnanolone acts to exert its suppressive effects on HPA responses to IL-1␤. Allopregnanolone may prevent stimulation of CRH neurons in the PVN by noradrenaline (Patchev et al., 1994) or it may inhibit CRH neurons by modulating GABA inputs to the PVN by its actions on GABA A receptors. For example, GABA neurons in the peri-PVN area mediate glucocorticoid negative feedback (Di et al., 2009), whereas GABA neurons in the bed nucleus of stria terminalis (Cullinan et al., 2008) can modulate HPA responses to IL-1␤ (Crane et al., 2003).
The NTS contains intrinsic GABA neurons (Fong et al., 2005;Bailey et al., 2008), GABA-containing terminals, and GABA A receptor subunits (Terai et al., 1998;Saha et al., 2001), and GABA release in the NTS is increased by depolarization (Sved and Curtis, 1993). Effects of GABA on NTS neurons identified as projecting to the PVN are not reported, nonetheless GABA inhibits the electrical activity of A2 neurons (Moore and Guyenet, 1983) and Figure 6. Effect of allopregnanolone treatment on induction of inhibitory opioid tone over HPA axis responses to IL-1␤ in virgin rats. Virgin rats were treated with allopregnanolone (3 and 1 mg/kg, s.c., 20 and 2 h before IL-1␤, respectively) or vehicle (oil). After a basal (B) blood sample (at t ϭ Ϫ31 min), rats were administered naloxone (NLX; 5 mg/kg, i.v.) or vehicle [saline (sal); 0.5 ml/kg, i.v.]. Two additional blood samples were collected at Ϫ16 and Ϫ1 min, after which all rats were administered IL-1␤ (500 ng/kg, i.v.) at t ϭ 0 min. Additional blood samples were taken 15, 30, 60, 90, and 120 min after treatment. Trunk blood was collected at t ϭ 240 min. a, b, Plasma ACTH concentration (a) and increase in ACTH secretion (b) from post-naloxone levels 15 min after IL-1␤. n ϭ 8 -10 rats per group. a, *p Ͻ 0.05 versus basal values in the same group; # p Ͻ 0.04 versus AP/sal group; b, *p Ͻ 0.05 versus AP/sal group. Rats were killed 4 h after IL-1␤ administration, and brains were processed by in situ hybridization. c, CRH mRNA expression in the pPVN. Quantification by in situ hybridization 4 h after intravenous IL-1␤, expressed as number of neurons expressing CRH mRNA per unit area pPVN. n ϭ 8 -13 rats per group. *p Ͻ 0.04 versus AP/saline group. All values are group means Ϯ SEM.

Figure 7.
Effect of allopregnanolone and finasteride on opioid mRNA expression in NTS. a, pENK-A mRNA expression in the A2 region of the NTS in virgin rats treated with AP (3 and 1 mg/kg, s.c.) or vehicle (veh) at t ϭ Ϫ20 and Ϫ2 h. Brains were collected at t ϭ 0 h. *p Ͻ 0.001 versus vehicle group. Grain density per NTS [in arbitrary units (a.u.)] is the number of positive cells per NTS section times grain density per cell. b, Representative photomicrographs of pENK-A mRNA in the A2 region of the NTS from vehicle-treated virgin (i) and AP-treated virgin (ii) rats. APo, Area postrema; CC, central canal; 10, dorsal motor nucleus of vagus; 12, hypoglossal nucleus. Scale bars: top, 100 m; bottom, 25 m. Vehicle-treated virgin (iii) and AP-treated virgin (iv) rats; higher-magnification views are indicated by the dashed box. c, pENK-A mRNA expression in the A2 region of the NTS in day 21 pregnantratstreatedwithfinasteride(FIN;25mg/kg,s.c.)orvehicle(veh)attϭϪ20andϪ2h.Brainswerecollectedattϭ0h.*pϽ 0.01 versus veh group. Grain density was calculated as above. n ϭ 5-7 rats per group. All values are group means Ϯ SEM.