Gender differences in the regulation of 3α-hydroxysteroid dehydrogenase in rat brain and sensitivity to neurosteroid-mediated stress protection

Neuroscience 120 (2003) 541–549

GENDER DIFFERENCES IN THE REGULATION OF 3␣HYDROXYSTEROID DEHYDROGENASE IN RAT BRAIN AND SENSITIVITY TO NEUROSTEROID-MEDIATED STRESS PROTECTION Y. A. MITEV,a1 M. DARWISH,b1 S. S. WOLF,a F. HOLSBOER,b O. F. X. ALMEIDAb AND V. K. PATCHEVa*

ation of neuroactive steroids, represented by allopregnanolone tetrahydroprogesterone (THP) and tetrahydrodeoxycorticosterone, respectively. Ring-A-reduction of PROG and CORT results in a graded loss in their ability to interact with the corresponding nuclear hormone receptors and a concomitant increase in their affinity for the (membrane-bound) steroid binding site of the receptor gamma amino butyric acid (GABA) (Paul and Purdy, 1992). 5␣,3␣-Hydroxylated steroids are now recognized as efficacious endogenous modulators of GABA-ergic neurotransmission, and anomalous biosynthesis of these compounds has been implicated in behavioral and neuroendocrine dysfunction, including the pathogenesis of neurological and psychiatric conditions (Rupprecht, 1997). During development, the brain is ‘organized’ and ‘activated’ in terms of morphology, chemistry and responsiveness to sensory modalities and endocrine signals as a result of programmed fluctuations in hormonal secretions, thus allowing differential adaptation to the external environment and milieu interieur. In view of evidence that treatment with ring-A-reduced steroids during early brain development can influence brain function in adulthood (Patchev et al., 1997), and because sex steroids can profoundly affect various aspects of physiological and behavioral adaptation throughout life (Patchev and Almeida, 1998), this study set out to map the regulation of the expression of 3␣-HSD in the rat brain throughout development and with respect to both gender and specific steroids of gonadal and adrenal origin. A further intention was to examine whether manifestation of previously described behavioral consequences of adverse events in early life and their attenuation by neonatal neurosteroid treatment (Patchev et al., 1997) depends on the gender and gonadal status of the adult individual. Thus, by combining different experimental approaches, we provide neuromorphological and behavioral data to show that (i) both, biosynthesis of, and responsiveness to neurosteroids are early-onset features of the rat brain, and correlate with distinct ontogenic phases of organism’s response to stress; (ii) sex hormones differentially control neurosteroid biosynthesis, a phenomenon which is already established during early development, and these sex hormone-dependent programs can be demonstrated through both differential behavioral liability and sensitivity to hormonal regulation; (iii) the sex-specific endocrine background and physiological gonadal secretions influence the manifestation of behavioral phenomena which are subject to neurosteroid control.

a Male Health Care II, Schering AG/Jenapharm, Otto Schott Strasse 15, 07745 Jena, Germany b

Max Planck Institute of Psychiatry, 80804 Munich, Germany

Abstract—The enzyme 3␣-hydroxysteroid dehydrogenase (3␣-HSD) is involved in the generation of neuroactive steroids through ring-A-reduction of hormonal precursors. We examined the developmental regulation of, gender differences in, and effects of hormonal manipulations on the expression of 3␣-HSD in the rat hippocampus. High levels of 3␣-HSD mRNA were found on postnatal day 7, coinciding with the stress hyporesponsive period in the rat. Gender differences in 3␣-HSD expression were documented during puberty, but not in adulthood. Adrenalectomy and gonadectomy, and supplementation with individual steroid hormones influenced 3␣-HSD expression in a gender-specific mode. We also demonstrate that the manifestation of behavioral and endocrine consequences of early life stress depends on the individual’s gender and gonadal status. Males are liable to aftereffects of neonatal maternal deprivation, regardless of their adult gonadal status. In females, however, anxiogenic aftereffects of neonatal stress become apparent only after gonadectomy. These data suggest that (i) transient increase of neurosteroid biosynthesis may contribute to stress hyporesponsiveness during early infancy; (ii) gonadal steroids regulate 3␣-HSD expression in the hippocampus in a sexspecific mode; (iii) physiological sex steroid secretions in females may mask behavioral consequences of adverse early life events, and (iv) concomitant treatment with the neurosteroid THP counteracts behavioral and endocrine dysregulation induced by neonatal stress in both genders. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: neurosteroid biosynthesis, sex differentiation, development, neonatal stress, steroid hormones.

The enzyme 3␣-hydroxysteroid dehydrogenase (3␣-HSD) is expressed in several mammalian tissues (Penning et al., 1996). Its multiple roles include ring-Areduction at the 3␣ position of progesterone (PROG) and corticosterone (CORT) with the subsequent gener1

These authors contributed equally to this work. *Corresponding author. Tel: ⫹49-3641-646211; fax: ⫹49-3641646085. E-mail address: [email protected]. (V. K. Patchev). Abbreviations: ADX, adrenalectomy; CORT, corticosterone; DG, dentate gyrus; DHT, dihydrotestosterone; E2, 17␤-estradiol; GABA, gamma amino butyric acid; GDX, gonadectomy; PROG, progesterone; SHRP, stress hyporesponsive period; THP, tetrahydroprogesterone, allopregnanolone; 3␣-HSD, 3␣-hydroxysteroid dehydrogenase.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00287-2

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EXPERIMENTAL PROCEDURES All experimental procedures were performed in compliance with national regulations and NIH guidelines on animal welfare. All efforts were made to minimize the number of animals and their suffering. Each experimental group included four to six animals.

3␣-HSD mRNA expression profiles Pregnant Wistar rat dams were purchased from Charles River (Sulzfeld, Germany). Animals were housed under controlled illumination (12-h light/dark, lights on at 7:00 a.m.) and temperature (23–24 °C), and had free access to food and water. Within 24 h of delivery, litters were culled to six pups. At the age of 21 days, pups were weaned and housed in sex-matched groups of five for the rest of the experiment. For the examination of developmental changes in the parameter of interest, rats were killed by decapitation at the age of 1, 7, 21, 40 and 90 days. The intermediate points of investigation between birth and sexual maturity (90 days) were selected because: (i) infant rats aged 5–15 days fail to display endocrine responses to stress (stress-hyporesponsive period; Sapolsky and Meaney, 1986); (ii) adult-like hippocampal architecture and responsiveness to glucocorticoids are well established at weaning age (21 days; Rosenfeld et al., 1993); (iii) puberty in the rat occurs between 35 and 40 days of age. Timing of puberty in female rats was monitored by checking the vaginal opening and occurrence of cyclic changes in vaginal epithelium (microscopic examination of daily vaginal smears taken between days 35 and 45). For developmental studies, animals were killed by decapitation at the following ages: day 1 (within 24 h of birth), day 7, day 21, days 40 – 45 (average age of puberty) and day 90 (sexual maturity). Pubertal and adult females were killed in diestrus. Gonadectomy (GDX) and adrenalectomy (ADX) were performed in adult animals under barbiturate anesthesia (Brevimytal; Lilly, Bad Homburg, Germany; 30 mg/kg i.p.), and animals were allowed to recover for 1 week before experimentation. Adrenalectomized animals were provided with physiological saline as drinking solution. For the examination of the effect of individual steroid hormones (all from Sigma-Aldrich, Deisenhofen, Germany), steroid-deprived rats (GDX plus ADX) received daily s.c. injections of either 10 ␮g 17␤-estradiol (E2), 100 ␮g dihydrotestosterone (DHT), 1 mg CORT or 5 mg PROG per 1 kg body weight for 3 consecutive days. Control animals were treated with vehicle (sesame oil). Mapping of the distribution of 3␣-HSD mRNA was done in serial coronal brain cryosections (15 ␮m) from treatment-naive neonatal (ages 1 and 7 days), juvenile (aged 21 days) and pubertal (aged 40 days) male and female rats and intact, ADX, ADX/ GDX and steroid-replaced ADX/GDX rats of both sexes. Sections were mounted onto gelatin-coated slides, followed by post-fixation, permeabilization and hybridization with an 35S-labeled cRNA probe. The latter was prepared from a plasmid expressing the full-length cDNA encoding the rat liver 3␣-HSD (generously provided by Dr. T. Penning, University of Pennsylvania School of Medicine, Philadelphia, PA, USA). 35S-labeled antisense and sense cRNAs were generated from the linearized plasmid (SspI and SalI) by in vitro transcription from the SP6 and T7 promoter, respectively. To enhance cellular penetration, the probes were fragmented by alkaline hydrolysis. Specificity of hybridization signals was monitored in sections pre-treated with RNA-se or incubated with labeled sense probe under identical conditions. Highstringency washes (60 min in 2⫻ SSC buffer at 50°C, and two consecutive washes in 0.2⫻ SSC at 55°C and 60°C for 60 min each) and other details of the protocol followed, as well as methods of semi-quantitative autoradiography, are described elsewhere (Whitfield et al., 1990). Quantification of hybridization signals, and age-, sex- and treatment-related comparisons were made in the dentate gyrus (DG), a subfield of the hippocampal formation displaying receptors for most adrenal and gonadal ste-

roid receptors, as well as high densities of 3␣-HSD-encoding transcripts (see below). In adult animals, the level of sectioning corresponded to bregma ⫺3.14 mm (Paxinos and Watson, 1986); in newborn and juvenile rats, the corresponding area was selected from serial sections using the medial extension of the hypothalamic ventromedial nucleus as a reference point. Film autoradiographs emerging from two adjacent sections were evaluated for each animal. Using the image analysis software NIH Image 1.52, integrated pixel densities were measured within the anatomical borders of the brain area of interest; these were subsequently converted to specific radioactivity by a polynomial equation derived from co-exposed radioactive standards. For each animal, an individual mean value was calculated from the four measurements described above and used for subsequent group statistics (Konakchieva et al., 1998). The investigator was aware of the age, but not sex or treatment, of the tissue donors.

THP-induced anxiolysis: role of gender and effects of GDX Pregnant female Wistar rats (day 16 of pregnancy) were obtained from Charles River (Sulzfeld, Germany) and maintained in our animal colony under the conditions mentioned above. Maternal separation of juvenile animals and neurosteroid administration was performed daily for 2 hours between postnatal days 5 and 10, as previously described (Patchev et al., 1997). THP (5␣-pregnan3␣-ol, 20-one; Steraloids, Newport, RI, USA) was injected s.c. at a daily dose of 2 mg/kg b.w. immediately prior to removal of the pups from the home cage. Control animals received vehicle (sesame oil containing 0.25% ethanol) injections. Subsets of maternally deprived (neonatally stressed) and control rats (⫾THP) were gonadectomized under halothane anesthesia at 60 days of age. After a 7 day period of recovery from surgery, all animals were tested for their level of anxiety in the plus-maze test (Dawson and Tricklebank, 1995). All intact females used for this part of the study were randomly cycling, i.e. not in a particular phase of the estrous cycle.

Effects of THP on stress-induced CORT secretion Male and female rat pups born in our animal colony (see above) were subjected to a single period of maternal deprivation (12 h between postnatal days 5 and 6) after which they were reunited with their mothers. One half of the animals of each gender received a single s.c. injection of THP (2 mg/kg dissolved in 37% 2-isopropyl-␤-cyclodextrin (Sigma-Aldrich, Deisenhofer, Germany) in water) immediately before separation from their mothers; the rest of the animals received a vehicle injection. When pups were 10 days old, those that had been previously subjected to maternal deprivation were given a further stress by placement with a non-lactating foster mother for 1 h, whereas the other animals were left undisturbed with their own mothers. One hour later, one half of all unstressed and stressed males and females were killed and their trunk blood collected for estimation of serum CORT levels using a commercial radioimmunoassay kit (ICN Biomedical, Costa Mesa, CA). The remaining pups were left with their mothers until weaning (day 21) and allowed to develop to adulthood (day 65) when their serum CORT levels were measured in trunk blood 30 min after brief exposure to emotional stress (intermittent air puffs for 2 min), as described elsewhere (Patchev et al., 1997). Females used in this experiment were randomly cycling.

Statistics Group means were compared by parametric (hybridization signals, hormone levels) or non-parametric (behavioral data) oneway ANOVA, followed by the Student-Newmann-Keuls or MannWhitney test, where appropriate. The level of significance was pre-set at P⬍0.05.

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clear complex and the isocortex at several levels of sectioning (frontal, parietal, temporal, occipital; in the latter structure, there were no obvious differences in signal abundance between individual anatomic regions or cytoarchitectonic layers. Faint signal densities were observed in the preoptic area and hypothalamic supraoptic, ventromedial and arcuate nuclei, while no transcripts could be detected in the anterior olfactory, suprachiasmatic, septal, pontine and lower brainstem nuclei, striatum and periaqueductal gray matter. Developmental and sex-specific changes in 3␣-HSD expression in the DG The DG exhibited the highest density of 3␣-HSD-encoding transcripts also in the neonatal rat brain. In both sexes, signal density significantly increased at postnatal day 7; subsequently, gradual decline was seen with increasing age. Signal density in adult rats was indistinguishable from that measured in neonates. Significant gender difference was documented only at puberty, with female animals displaying significantly higher transcript densities than males. At all other time point examined, hybridization signal densities in the DG were similar in both sexes (Fig. 2). Effect of GDX and ADX on 3␣-HSD expression in the adult DG While mRNA levels measured in intact males and diestrous females were similar, deprivation of gonadal or adrenal steroids differentially affected hybridization signal density in both sexes. ADX was associated with significant decreases in transcript densities in males and females; however, GDX reduced 3␣-HSD expression only in the male DG (Fig. 3). Effect of steroid hormone replacement on 3␣-HSD expression in the adult DG Fig. 1. Distribution of 3␣-HSD-encoding transcripts at selected coronal levels of the adult rat brain.

RESULTS Regional distribution of 3␣-HSD mRNA in the adult rat brain Strong hybridization signals were documented in the hippocampal formation, especially in the DG, the islands of Calleja in the olfactory lobe, and the cerebellar cortex (Fig. 1). Moderate signal intensity was seen in the hypothalamic paraventricular nucleus, habenular nuclei, amygdaloid nu-

Administration of gonadal or adrenal steroids in animals completely deprived of endogenous steroid hormones influenced the density of 3␣-HSD-encoding transcripts in a gender-specific fashion. Steady-state mRNA levels were similar in male and female ADX/GDX rats receiving placebo. Administration of CORT resulted in significant increases in signal density in both sexes. Treatment with E2 significantly augmented 3␣-HSD mRNA levels in the female, but not male, hippocampus. Administration of the pure androgen DHT up-regulated 3␣-HSD expression in the DG of male, but not female, rats. PROG administration failed to significantly influence hybridization signal density in both sexes (Fig. 4).

Abbreviations used in the figures CA hippocampal subfield CA1–2 CI Calleja islands CON intact control

MS

maternal separation

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Fig. 2. Age and gender profiles of 3␣-HSD expression in the rat DG. Asterisks indicate significant differences compared with newborn animals of the same sex; daggers denote significant gender differences within the same age group. Each bar represents mean⫾S.E.M. of four to five individuals.

Analysis of the role of gender and effects of GDX on THP-induced anxiolysis As reflected by the time spent in the closed arm of the elevated plus maze, untreated testis-intact non-stressed male rats displayed significantly higher anxiety than their untreated ovary-intact non-stressed female counterparts. Maternal separation stress during early postnatal development resulted in significantly increased anxiety measured in adulthood in males. The impact of GDX in adulthood on anxiety in males was not significant, regardless of their history of neonatal stress. Since basal anxiety levels in males are high, this finding probably reflects a ‘ceiling effect’ of the methodology. Neonatal administration of THP significantly reduced the level of anxiety in males, independent of their gonadal status (Fig. 5A). Maternal separation during infancy failed to significantly alter adult anxiety levels in gonad-intact female rats; however, GDX in adulthood resulted in strong increase in anxiety in neonatally stressed females. Concomitant administration of THP in females subjected to maternal separation during infancy significantly attenuated manifestation of GDX-induced anxiety in adulthood. The neurosteroid alone did not exert any anxiolytic effects in intact female rats (Fig. 5B). Ability of THP to attenuate perinatal stressassociated increases in CORT secretion: gender specificity Pups aged 10 days failed to display a secretory response to stress inflicted by brief exposure to a non-lactating foster mother. However, previous maternal separation for 12 h on postnatal days 5– 6 elicited significant CORT increases in

male and female pups; at this age, no significant gender differences in the stress response were present. A single injection of THP at the time of maternal separation significantly attenuated the acute stress-induced CORT increase in male, but not female, pups. THP alone failed to significantly influence acute stress-induced CORT secretion in rats which were not exposed to maternal separation (Fig. 6A). The adrenal secretory response of adult rats to brief emotional stress showed significant differences related to gender and neonatal stress history: as previously reported, stress-induced CORT levels were higher in females than males (Patchev et al., 1998), and neonatal adverse experience was associated with exaggerated secretory responses in both genders (Patchev et al., 1997). Neonatal THP administration alone failed to influence CORT levels, but significantly attenuated or abolished excessive secretory responses in neonatally stressed males and females, respectively (Fig. 6B).

DISCUSSION The enzyme 3␣-HSD plays a key role in the biosynthesis of ring-A-reduced steroids which, through their interactions with an allosteric binding site on the GABAA receptor, serve as endogenous sedatives and stress-protectors. Here, we confirm previous work showing that 3␣-HSD mRNA maps to rat brain areas enriched in GABAA binding sites, namely, the hippocampus, cerebellum and cortex (Krieger and Scott, 1989; Khanna et al., 1995; Li et al., 1997; Mensah-Nyagan et al., 1999). Complementing recent studies showing that 3␣-HSD activity in the hypothalamus (Gao et al., 2002) and allopregnanolone (THP) lev-

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Fig. 3. Effects of partial (either ADX or GDX) and complete (ADX⫹GDX) steroid hormone deprivation on 3␣-HSD transcript densities in the DG. Asterisks indicate significant differences when compared with the sex-matched CON group; daggers denote significant gender differences in response to a given treatment. Each value represents mean⫾S.E.M. of four to five individuals.

els in the cerebral cortex (Grobin and Morrow, 2001) vary with age, we now describe ontogenetic and gender-specific patterns of 3␣-HSD mRNA distribution in these areas. Further, we demonstrate that these developmental and gender profiles are influenced by gonadal and adrenal steroid secretions, and that together, all these factors contribute to gender differences in the ability of the neurosteroid THP to influence anxiety and the hormonal response to stress. Here, we focused on alterations in 3␣-HSD mRNA levels in the hippocampus since this brain region is especially important for the neural regulation of the endocrine and behavioral response to stress. In addition, the involvement of the hippocampus, a major component of the limbic circuit, in the modulation of emotional state by corticosteroids (Korte, 2001) and PROG metabolites (Rhodes and Frye, 2001) has been strongly suggested, although not conclusively established. In intact animals, 3␣-HSD-encoding transcript levels in the hippocampus remained relatively constant from birth through to adulthood except at 7 days of age when a significant increase was found in both males and females. In the rat, a so-called stress-hyporesponsive period (SHRP) has been described during the first 2 postnatal weeks (Sapolsky and Meaney, 1986). Thus, the observed increase of 3␣-HSD mRNA expression during the SHRP suggests that elevated local biosynthesis of neurosteroids may contribute to a larger complex of mechanisms governing the organism’s response to stress. In light of previous observations, including our own (Patchev et al., 1997), that neurosteroids provide long-term protection against the adverse effects of early life stressful events, the present finding strongly suggest a role for neurosteroids in attenuating the vulnerability of the developing brain to the det-

rimental consequences of stress. The latter include increased anxiety and hypersecretion of adrenocortical hormones during adulthood. Addressing the question of gender-related differences in the expression of 3␣-HSD mRNA in the hippocampus, we found that such differences are measurable only around the time of puberty, when female rats expressed significantly higher levels of 3␣-HSD mRNA. This finding most probably reflects a sexually differentiated response, resulting from perinatal sexual differentiation of relevant neural substrates and the ‘activational’ effects of estrogens, the secretion of which increases in females at the onset of puberty (Ojeda and Urbanski, 1994). Indeed, as discussed below, hippocampal 3␣-HSD mRNA levels are up-regulated by E2 and changes in the gonadal steroid milieu during adulthood exert an influence over neurosteroid-sensitive physiological and behavioral processes. Given the cyclic pattern of E2 secretion in the female, it will be important to know whether 3␣-HSD mRNA expression also occurs in a cyclical fashion; such information could find application in the therapeutic context especially since a recent study has shown that the anxioyltic potency of THP varies according to the phase of the rat estrous cycle (Laconi et al., 2001). Previous studies yielded equivocal interpretations with respect to the role of endogenous steroids in the regulation of 3␣-HSD expression (Li et al., 1997; Cheng et al., 1994; Stoffel-Wagner et al., 2000). Here, selective deprivation of either gonadal or adrenal steroids in adult animals revealed significant influences of individual steroid classes on the constitutive expression of 3␣-HSD mRNA in the hippocampus. GDX in adult male, but not female, rats resulted in a significant decrease in the levels of 3␣-HSD in

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Fig. 4. Effects of supplementation with steroid hormones on 3␣-HSD mRNA levels in the DG of rats deprived of endogenous steroids (GDX/ADX). Asterisks indicate significant differences to placebo-treated GDX/ADX animals (mean⫾S.E.M. represented by shaded areas); each bar represents mean⫾S.E.M. of five individuals.

the hippocampus. Supplementation of steroid-free (ADX/ GDX) animals with sex-characteristic gonadal steroids revealed that the androgen DHT acted to restore basal 3␣HSD mRNA expression in males, whereas females only responded to E2, i.e. 3␣-HSD mRNA levels are regulated in each gender only by gender-specific sex steroid. Interestingly, PROG, a major substrate of 3␣-HSD, did not influence the expression of 3␣-HSD mRNA in either gender. Elimination of adrenocortical secretions by ADX also resulted in a significant lowering of 3␣-HSD mRNA levels in adult male and female rat hippocampi. These data confer with those reported for the male rat from Penning’s laboratory (Hou et al., 1998). Corroboration that adrenocortical steroids play a leading role in the regulation of 3␣-HSD gene transcription in both sexes was provided by data obtained in ADX/GDX animals that were given CORT substitution therapy. Animals of both sexes responded with increased hippocampal 3␣-HSD mRNA levels that were significantly higher than those found after ADX and similar to levels found in intact animals. Together with previous reports that cerebral neurosteroid synthesis can be induced by stress and/or increased corticosteroid secretion, the present data and our earlier findings that neurosteroids attenuate the consequences of stress on emotional state as well as short (Patchev et al., 1996) and long-term (Patchev et al., 1997) hormonal responses to stress add considerable weight to the hypothesis that neurosteroids play an important role in the neurochemical control of behavioral and secretory responses to stress. With regard to the latter, we submit that increased neurosteroid production and action(s) in the hippocampus and/or hypothalamus trigger homeostatic adjustments, thus facilitating appropriate curtailment of the corticosteroid response to

stress (Paul and Purdy, 1992; Patchev et al., 1996, 1997; Rupprecht, 1997; Purdy et al., 1991). Based on the conclusion that 3␣-HSD mRNA levels are stimulated by adrenocorticoids, androgens and estrogens, it is conceivable that diminished secretion of these steroids during different pathological conditions (e.g. hypogonadism, adrenocortical insufficiency) and phases of life (menopause, andropause) will be accompanied by reduced cerebral production of neurosteroids, with likely consequences for behavioral and physiological regulatory mechanisms. We previously reported that THP treatment attenuated ADX-induced dentate granule cell losses in the hippocampus (Conde´ et al., 1998), a finding which extends the notion that reduced neurosteroid availability can have deleterious consequences in the brain. As already mentioned, the stress-protective and anxiolytic actions of neurosteroids are well known (Paul and Purdy, 1992; Rupprecht, 1997; Purdy et al., 1991). To complement the information on hormonal regulation of 3␣-HSD expression, we also undertook studies to examine how the long-term consequences (increased anxiety and hypersecretion of CORT in adulthood) of stress inflicted during the neonatal period, and ameliorating effects of concomitant neurosteroid administration, are influenced by the individual’s gender and gonadal status. To aid interpretation of the results, it is important to note that (i) THP and stress were administered daily between days 5 and 10 or on days 5– 6, thus overlapping with the stress hyporesponsive period and a time when hippocampal levels of 3␣-HSD mRNA reach a peak, and (ii) that females display lower basal levels of anxiety as compared with males (Johnston and File, 1991; Bitran and Dowd, 1996; DiazVeliz et al., 1997). Also, it is important to record that the

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Fig. 5. Comparison of anxiolytic actions of neonatally administered THP in adult male (panel A) and female rats (panel B) that had been exposed to repeated MS during infancy, and undergone GDX (solid bars) or sham-operation (open bars) as adults. Asterisks indicate significant effects of neonatal treatment, as compared with control gonad-intact or gonadectomized animals without neonatal stress exposure (CON). Daggers denote significant effect of GDX in groups receiving identical neonatal treatment; triangles indicate significant anxiolytic effect of neonatal THP administration, as compared with the corresponding MS groups. Data depicted are means⫾S.E.M. (n⫽6 rats per group).

females used for these behavioral measurements were randomly cycling to avoid cycle-dependent bias. The present data show that behavioral dysregulation resulting from neonatal stress is present in adult males regardless of their gonadal status, whereas in females these consequences become manifest only after GDX. This observation strongly suggests that physiological gonadal secretions in the female can mask behavioral pathology resulting from adverse early life experience, with symptoms emerging only after cessation of ovarian secretory activity. With respect to neurosteroid biosynthesis, it remains to be clarified whether ovariectomy-induced anxiety results from deprivation of PROG, a major precursor of neurosteroids, or lack of estrogen-mediated induction of 3␣-HSD in the brain. A role for neurosteroids in the longterm control of behavioral and endocrine responsiveness to stress is corroborated by the demonstration that THP administration concomitantly with neonatal stress counteracts symptoms of dysregulation in infancy (CORT response to acute stress in males) and adulthood. Interestingly, THP also acted as an effective anxiolytic in neonatal stress-naive male rats, and attenuated exaggerated secretory responsiveness to stress in infant males. This finding supports the view that sensitivity to THP is a sexually differentiated event, and tempts to speculate that males are more liable to long-term consequences of adverse early life experience, but also more sensitive to neurosteroid-mediated neuroprotection, with gonadal secretions playing rather a subordinate role. On the other hand, dependence of symptom manifestation on the gonadal status

in females, and influence of estradiol on 3␣-HSD expression suggests that gonadal steroids in the female may significantly contribute to the neurosteroid-mediated control of behavior and pituitary-adrenal secretions. Further investigations, with greater focus on the interactions between gonadal steroids and neurosteroid availability, are required to resolve the mechanistic basis for, and the therapeutic implications of, these observations. In view of the presently-reported lack of effects of PROG to influence 3␣-HSD mRNA expression and, at the same time, a clinical study showing that PROG can potentiate sensitivity to neurosteroids in postmenopausal women (Wihlback et al., 2001), studies on the role of PROG in the modulation of 3␣-HSD activity and/or the pharmacological properties of GABAA receptor are also warranted. Specific consideration also deserves 5␣-reductase, an enzyme with a critical role for the initiation of the ring-A-reduction of steroids. This enzyme is present and functional in the brain, especially during early development (Lauber and Lichtensteiger, 1996; Melcangi et al., 1998; Kellogg and Frye, 1999; Stoffel-Wagner 2001). Investigations on the regulation of, and sex differences in the expression of 5␣-reductase in the brain have focused on the hypothalamus; albeit controversial, evidence for sex-specific hormonal control (Karolczak et al., 1998; Gao et al., 2002; Matsui et al., 2002) and distinct developmental profiles of this enzyme’s expression and activity (Kellogg and Frye, 1999; Eechaute et al., 1999) merit further attention, also in the context of these results.

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Fig. 6. Effects of single neonatal THP treatment on acute stress-induced CORT secretion in juvenile (10 day old, panel A) and adult (panel B) rats that had been exposed to MS for 12 h at the age of 5– 6 days. THP was administered prior to maternal separation; exposure to a non-lactating foster mother or intermittent air puffs served as acute stressors in juvenile and adult animals, respectively. Asterisks indicate significant differences, as compared with animals without MS experience; triangles denote significant CORT-lowering effect of THP, as compared with MS alone. Data depicted are means⫾S.E.M. (n⫽6 rats per group).

In summary, these results suggest that neurosteroids may play a critical role in determining the liability of the neonatal brain to adverse consequences of early life stress in both genders, with males displaying stronger sensitivity to the protective action of exogenous neurosteroids. In the female, physiological gonadal secretions may prevent the manifestation of symptoms of behavioral end neuroendocrine impairment resulting from neonatal stress; together with evidence for sex-specific regulation of neurosteroid biosynthesis, these data indicate a critical involvement of ovarian steroids in neurosteroid signaling in the female brain. Acknowledgements—These studies were supported by the German Federal Ministry of Education and Research (grant 0311469A). We are grateful to Dr. T. Penning, University of Pennsylvania School of Medicine, Philadelphia, PA, USA for the gift of rat 3␣-HSD cDNA. Support from the Stanley Foundation (O.F.X.A.) is gratefully acknowledged; M.D. was the recipient of a pre-doctoral training grant from the German Academic Exchange Service (DAAD).

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(Accepted 4 April 2003)