Motivated behaviors and levels of 3α,5α-THP in the midbrain are attenuated by knocking down expression of pregnane xenobiotic receptor in the midbrain ventral tegmental area of proestrous rats.

INTRODUCTION: Progesterone (P4 ) and its product, 5α-pregnan-3α-ol-20-one (3α,5α-THP), act in the midbrain ventral tegmental area (VTA) to alter motivated behaviors, such as mating, and motor and anxiety behavior. Of interest is whether 3α,5α-THP formation requires the pregnane xenobiotic receptor (PXR), which is expressed in the midbrain of rats. AIM: The role of PXR in the midbrain for 3α,5α-THP formation, which precedes modulation of motivated behaviors, was investigated. METHODS: Rats had estrous cycle phase determined and were assessed when they were in diestrus or proestrus. Diestrous and proestrous rats were infused with control or antisense oligodeoxyribonucleotides (AS-ODNs) targeted against PXR to the VTA. MAIN OUTCOME MEASURES: In pilot studies, PXR gene and protein expression in the midbrain were determined with quantitative reverse transcriptase polymerase chain reaction and Western blotting, respectively. Diestrous and proestrous rats infused with control or AS-ODNs to the VTA were tested for anxiety (open field and plus maze), social (social interaction), and sexual (paced mating) behavior. Expression of PXR in the midbrain was verified with Western blotting. Plasma estradiol, P4 , dihydroprogesterone (DHP), and 3α,5α-THP levels, and brain P4 , DHP, and 3α,5α-THP levels were measured. We predicted that proestrous rats infused with PXR AS-ODNs would have decreased anti-anxiety, social, and sexual behavior, lower midbrain expression of PXR, and lower midbrain levels of 3α,5α-THP compared with controls. RESULTS: Results supported the hypothesis that formation of 3α,5α-THP requires PXR and may be important for motivated behaviors. PXR AS-ODN, compared with control, infusions to the VTA reduced PXR expression and 3α,5α-THP levels in the midbrain and attenuated sexual receptivity of proestrous rats. CONCLUSIONS: Knockdown of PXR in the midbrain reduces 3α,5α-THP levels and sexual receptivity of proestrous rats. Thus, PXR in the midbrain may be required for the observed increase in 3α-5α-THP during proestrus, which has subsequent effects on motivated, reproductive behaviors.

The Steroidogenesis Inhibitor Finasteride Reduces the Response to Both Stressful and Rewarding Stimuli.

Finasteride (FIN) is the prototypical inhibitor of steroid 5α-reductase (5αR), the enzyme that catalyzes the rate-limiting step of the conversion of progesterone and testosterone into their main neuroactive metabolites. FIN is clinically approved for the treatment of benign prostatic hyperplasia and male baldness; while often well-tolerated, FIN has also been shown to cause or exacerbate psychological problems in vulnerable subjects. Evidence on the psychological effects of FIN, however, remains controversial, in view of inconsistent clinical reports. Here, we tested the effects of FIN in a battery of tests aimed at capturing complementary aspects of mood regulation and stress reactivity in rats. FIN reduced exploratory, incentive, prosocial, and risk-taking behavior; furthermore, it decreased stress coping, as revealed by increased immobility in the forced-swim test (FST). This last effect was also observed in female and orchiectomized male rats, suggesting that the mechanism of action of FIN does not primarily reflect changes in gonadal steroids. The effects of FIN on FST responses were associated with a dramatic decrease in corticotropin release hormone (CRH) mRNA and adrenocorticotropic hormone (ACTH) levels. These results suggest that FIN impairs stress reactivity and reduces behavioral activation and impulsive behavior by altering the function of the hypothalamus-pituitary-adrenal (HPA) axis.

Estradiol or diarylpropionitrile administration to wild type, but not estrogen receptor beta knockout, mice enhances performance in the object recognition and object placement tasks.

Cognitive processes mediated by the hippocampus and cortex are influenced by estradiol (E(2)); however, the mechanisms by which E(2) has these effects are not entirely clear. As such, studies were conducted to begin to address the role of actions at the beta form of the intracellular estrogen receptor (ERbeta) for E(2)'s cognitive effects in adult female mice. We investigated whether E(2) improved performance of wild type (WT) and ERbeta knockout (betaERKO) mice in tasks considered to be mediated by the cortex and hippocampus, the object recognition and object placement tasks. WT and betaERKO mice were ovariectomized (ovx) and E(2) (0.1 mg/kg), an ERbeta selective ER modulator (SERM), diarylpropionitrile (DPN; 0.1 mg/kg), or oil vehicle was administered to mice following training in these tasks. We hypothesized that if E(2) has mnemonic effects, in part, due to its actions at ERbeta, then WT mice administered E(2) or DPN would have improved performance compared to vehicle WT controls, which would not be different from betaERKO mice administered vehicle, E(2) or DPN. Alternatively, activation of ERalpha (with E(2), which is a ligand for both ERalpha and ERbeta) may produce opposing effects on cognition and/or the activation of ERalpha and ERbeta vs. either receptor isoform alone may produce a different pattern of effects. Results obtained supported the hypothesis that ERbeta activation is important for mnemonic effects. Ovx WT, but not betaERKO, mice administered E(2) or DPN had a greater percentage of time exploring a novel object in the object recognition task and a displaced object in the object placement task. Thus, actions at ERbeta may be important for E(2) or SERMs to enhance cognitive performance of female mice in the object recognition and placement tasks.

Estradiol or diarylpropionitrile decrease anxiety-like behavior of wildtype, but not estrogen receptor beta knockout, mice.

Clinical and basic studies demonstrate that estrogen (E-sub-2)-based therapies influence anxiety and mood, but the receptor targets (e.g., a or ss isoform of the estrogen receptor, ER) for these effects requires further investigation. To address the specificity of E2's anxiolytic-like effects through ERss, anxiety, motor, and nociceptive behavior of ovariectomized, wildtype (WT), and ERss knockout (ssERKO) mice was examined. Mice were administered oil vehicle or ER agonists, 17ss-E2 (E2; 0.1 mg/kg; similar affinity for ERa and ERss), and a selective ER modulator, diarylpropionitrile (DPN; 0.1 mg/kg; greater affinity for ERss than ERa). Performance of mice in anxiety (open field, elevated plus maze, elevated zero maze, social interaction), motor activity (activity monitor) and nociception (tailflick, pawlick) measures was compared. Results supported our hypothesis that ERss is important in modulation of anxiety-like behavior by E2 in some tasks. Administration of E2 or DPN to WT, but not ssERKO, mice increased open field central entries, plus maze open arm time, zero maze open quadrant time, and social interaction. This pattern was neither seen in motor activity nor pain threshold measures. Thus, actions of ERss may be important for modulating anxiety-like behavior of mice.

Androgens with activity at estrogen receptor beta have anxiolytic and cognitive-enhancing effects in male rats and mice.

Testosterone (T) and its metabolites may underlie some beneficial effects for anxiety and cognition, but the mechanisms for these effects are unclear. T is reduced to dihydrotestosterone (DHT), which can be converted to 5alpha-androstane,3alpha,17beta-diol (3alpha-diol) and/or 5alpha-androstane-3beta,17beta-diol (3beta-diol). Additionally, T can be converted to androstenedione, and then to androsterone. These metabolites bind with varying affinity to androgen receptors (ARs; T and DHT), estrogen receptors (ERbeta; 3alpha-diol, 3beta-diol), or GABA(A)/benzodiazepine receptors (GBRs; 3alpha-diol, androsterone). Three experiments were performed to investigate the hypothesis that reduced anxiety-like and enhanced cognitive performance may be due in part to actions of T metabolites at ERbeta. Experiment 1: Gonadectomized (GDX) wildtype and ERbeta knockout mice (betaERKO) were subcutaneously (SC) administered 3alpha-diol, 3beta-diol, androsterone, or oil vehicle at weekly intervals, and tested in anxiety tasks (open field, elevated plus maze, light-dark transition) or for cognitive performance in the object recognition task. Experiment 2: GDX rats were administered SC 3alpha-diol, 3beta-diol, androsterone, or oil vehicle, and tested in the same tasks. Experiment 3: GDX rats were androsterone- or vehicle-primed and administered an antagonist of ARs (flutamide), ERs (tamoxifen), or GBRs (flumazenil), or vehicle and then tested in the elevated plus maze. Both rats and wildtype mice, but not betaERKO mice, consistently had reduced anxiety and improved performance in the object recognition task. Androsterone was only effective at reducing anxiety-like behavior in the elevated plus maze and this effect was modestly reduced by flumazenil administration. Thus, actions at ERbeta may be required for T's anxiety-reducing and cognitive-enhancing effects.

Progestogens' effects and mechanisms for object recognition memory across the lifespan.

This review explores the effects of female reproductive hormones, estrogens and progestogens, with a focus on progesterone and allopregnanolone, on object memory. Progesterone and its metabolites, in particular allopregnanolone, exert various effects on both cognitive and non-mnemonic functions in females. The well-known object recognition task is a valuable experimental paradigm that can be used to determine the effects and mechanisms of progestogens for mnemonic effects across the lifespan, which will be discussed herein. In this task there is little test-decay when different objects are used as targets and baseline valance for objects is controlled. This allows repeated testing, within-subjects designs, and longitudinal assessments, which aid understanding of changes in hormonal milieu. Objects are not aversive or food-based, which are hormone-sensitive factors. This review focuses on published data from our laboratory, and others, using the object recognition task in rodents to assess the role and mechanisms of progestogens throughout the lifespan. Improvements in object recognition performance of rodents are often associated with higher hormone levels in the hippocampus and prefrontal cortex during natural cycles, with hormone replacement following ovariectomy in young animals, or with aging. The capacity for reversal of age- and reproductive senescence-related decline in cognitive performance, and changes in neural plasticity that may be dissociated from peripheral effects with such decline, are discussed. The focus here will be on the effects of brain-derived factors, such as the neurosteroid, allopregnanolone, and other hormones, for enhancing object recognition across the lifespan.

Female mice with deletion of Type One 5α-reductase have reduced reproductive responding during proestrus and after hormone-priming.

The capacity to form progesterone (P4)'s 5α-reduced metabolite, 5α-pregnan-3α-ol-20-one (3α,5α-THP; a.k.a. allopregnanolone), in the brain may be related to facilitation of lordosis among estrogen-primed (E2) mice. We investigated this idea further by comparing effects of endogenous and exogenous progestogens in mice that are deficient in the Type One 5α-reductase enzyme (5α-reductase knockout mice; 5α-RKO), and their wildtype counterparts for sexual behavior. Comparisons were made following administration of progestogens that are expected to increase 3α,5α-THP or not. Sexual receptivity of 5α-RKO mice and their wildtype counterparts was examined when mice were naturally-cycling (Experiment 1); ovariectomized (OVX), E2-primed (10 µg, subcutaneous; SC) and administered P4 (0, 125, 250, or 500 µg SC; Experiment 2); and OVX, E2-primed and administered P4, medroxyprogesterone acetate (MPA, 4 mg/kg, SC, which does not convert to 3α,5α-THP) or 3α,5α-THP (4 mg/kg, SC; Experiment 3). The percentage of mounts that elicited lordosis (lordosis quotient) or aggression/rejection behavior (aggression quotient), as well as the quality of lordosis (lordosis rating), was scored. Wildtype, but not 5α-RKO, mice in behavioral estrus demonstrated significantly greater lordosis quotients and lordosis ratings, but similar aggression quotients, compared to their diestrous counterparts. Among OVX and E2-primed mice, P4 facilitated lordosis of wildtype, but not 5α-RKO, mice. MPA neither facilitated lordosis of wildtype, nor 5α-RKO mice. 3α,5α-THP administered to wildtype or 5α-RKO mice increased lordosis quotients and lordosis ratings and decreased aggression quotients. 3α,5α-THP levels in the midbrain, one brain region important for sexual behavior, were increased during behavioral estrus, with P4 administered to WT, but not 5α-RKO mice, and 3α,5α-THP administered to WT and 5α-RKO mice. MPA did not increase 3α,5α-THP. Thus, deletion of Type One 5α-reductase among female mice may attenuate reproductive responding during the estrous cycle and after hormone-priming.

The pregnane xenobiotic receptor, a prominent liver factor, has actions in the midbrain for neurosteroid synthesis and behavioral/neural plasticity of female rats.

A novel factor of interest for growth/plasticity in the brain is pregnane xenobiotic receptor (PXR). PXR is a liver factor known for its role in xenobiotic clearance and cholesterol metabolism. It is expressed in the brain, suggesting a potential role for plasticity, particularly involving cholesterol-based steroids and neurosteroids. Mating induces synthesis of neurosteroids in the midbrain Ventral Tegmental Area (VTA) of female rodents, as well as other "plastic" regions of the brain, including the hippocampus, that may be involved in the consolidation of the mating experience. Reducing PXR in the VTA attenuates mating-induced biosynthesis of the neurosteroid, 5α-pregnan-3α-ol-20-one (3α,5α-THP). The 18 kDA translocator protein (TSPO) is one rate-limiting factor for 3α,5α-THP neurosteroidogenesis. The hypothesis tested was that PXR is an upstream factor of TSPO for neurosteroidogenesis of 3α,5α-THP in the VTA for lordosis, independent of peripheral glands. First, proestrous rats were administered a TSPO blocker (PK11195) and/or 3α,5α-THP following infusions of PXR antisense oligonucleotides (AS-ODNs) or vehicle to the VTA. Inhibiting TSPO with PK11195 reduced 3α,5α-THP levels in the midbrain and lordosis, an effect that could be reversed with 3α,5α-THP administration, but not AS-ODN+3α,5α-THP. Second, proestrous, ovariectomized (OVX), or ovariectomized/adrenalectomized (OVX/ADX) rats were infused with a TSPO enhancer (FGIN 1-27) subsequent to AS-ODNs or vehicle to the VTA. PXR AS-ODNs blocked actions of FGIN 1-27 for lordosis and 3α,5α-THP levels among proestrous > OVX > OVX/ADX rats. Thus, PXR may be upstream of TSPO, involved in neurosteroidogenesis of 3α,5α-THP in the brain for plasticity. This novel finding of a liver factor involved in behavioral/neural plasticity substantiates future studies investigating factors known for their prominent actions in the peripheral organs, such as the liver, for modulating brain function and its augmentation.

Novel receptor targets for production and action of allopregnanolone in the central nervous system: a focus on pregnane xenobiotic receptor.

Neurosteroids are cholesterol-based hormones that can be produced in the brain, independent of secretion from peripheral endocrine glands, such as the gonads and adrenals. A focus in our laboratory for over 25 years has been how production of the pregnane neurosteroid, allopregnanolone, is regulated and the novel (i.e., non steroid receptor) targets for steroid action for behavior. One endpoint of interest has been lordosis, the mating posture of female rodents. Allopregnanolone is necessary and sufficient for lordosis, and the brain circuitry underlying it, such as actions in the midbrain ventral tegmental area (VTA), has been well-characterized. Published and recent findings supporting a dynamic role of allopregnanolone are included in this review. First, contributions of ovarian and adrenal sources of precursors of allopregnanolone, and the requisite enzymatic actions for de novo production in the central nervous system will be discussed. Second, how allopregnanolone produced in the brain has actions on behavioral processes that are independent of binding to steroid receptors, but instead involve rapid modulatory actions via neurotransmitter targets (e.g., γ-amino butyric acid-GABA, N-methyl-D-aspartate- NMDA) will be reviewed. Third, a recent focus on characterizing the role of a promiscuous nuclear receptor, pregnane xenobiotic receptor (PXR), involved in cholesterol metabolism and expressed in the VTA, as a target for allopregnanolone and how this relates to both actions and production of allopregnanolone will be addressed. For example, allopregnanolone can bind PXR and knocking down expression of PXR in the midbrain VTA attenuates actions of allopregnanolone via NMDA and/or GABAA for lordosis. Our understanding of allopregnanolone's actions in the VTA for lordosis has been extended to reveal the role of allopregnanolone for broader, clinically-relevant questions, such as neurodevelopmental processes, neuropsychiatric disorders, epilepsy, and aging.

Progesterone facilitates exploration, affective and social behaviors among wildtype, but not 5α-reductase Type 1 mutant, mice.

Progesterone (P4) facilitates exploration, anxiety and social behaviors in estrogen (E2)-primed mice. Some of these effects may be due to actions of its 5α-reduced metabolite, 5α-pregnan-3α-ol-20-one (3α,5α-THP). In order to address the role of P4 and its metabolite, 3α,5α-THP, a mouse model was utilized. We hypothesized that if P4's metabolism to 3α,5α-THP is essential to facilitate exploratory, anti-anxiety and social behaviors of mice, then wildtype, but not 5α-reductase knockout (5α-RKO), mice will have greater expression of these behaviors. Experiment 1: Mice were ovariectomized (ovx), E2-primed and administered P4 (0, 125, 250, or 500µg) subcutaneously and then tested 4h later in a battery of tasks: open field, elevated plus maze, and social interaction. Experiment 2: Ovx, E2-primed mice were administered P4 (4mg/kg), 3α,5α-THP (4mg/kg), medroxyprogesterone acetate (MPA, which does not convert to 3α,5α-THP; 4mg/kg), or vehicle subcutaneously and tested 4h later. There was a dose-dependent effect of P4 to wildtype, but not 5α-RKO, mice. Neither wildtype, nor 5α-RKO, mice had increased exploration, anti-anxiety or pro-social behavior with MPA administration. Progesterone only exerted effects on anti-anxiety behavior, and increased 3α,5α-THP in the prefrontal cortex and hippocampus, when administered to wildtype mice. 3α,5α-THP to both WT and 5α-RKO mice increased exploration, anti-anxiety and social interaction and 3α,5α-THP levels in the hippocampus and prefrontal cortex. Thus, metabolism of P4 by the 5α-reductase enzyme may be essential for enhancement of these behaviors.

Progesterone, compared to medroxyprogesterone acetate, to C57BL/6, but not 5α-reductase mutant, mice enhances object recognition and placement memory and is associated with higher BDNF levels in the hippocampus and cortex.

Progesterone (P4) may influence cognition in part through actions of its 5α-reduced metabolite, allopregnanolone. Ovariectomized mice that were C57BL/6 wildtype (WT), or deficient in the 5α-reductase Type 1 enzyme (5α-reductase knockout; 5αRKO), were administered vehicle, P4, allopregnanolone, or medroxyprogesterone acetate (MPA) after training in the object recognition or placement tasks. WT mice administered P4 or allopregnanolone performed significantly better in the object recognition and placement tasks than did WT mice administered vehicle or MPA. 5αRKO mice administered allopregnanolone, but not P4, MPA, or vehicle showed enhanced performance in the object recognition and placement tasks. Levels of brain-derived neurotrophic factor (BDNF) in the prefrontal cortex and hippocampus were lowest among mice administered MPA. Thus, some of P4s effects to enhance cognitive performance may be incumbent upon its 5α-reduction.

Type 1 5α-reductase may be required for estrous cycle changes in affective behaviors of female mice.

There are estrous cycle differences in affective behaviors of rodents that are generally attributed to cyclic variations in estradiol, progesterone (P) and its metabolites. A question is the role of the steroid metabolism enzyme, 5α-reductase, for these estrous cycle differences. To address the requirement of 5α-reductase, estrous cycle variations in the behavior of wildtype mice and their littermates that are deficient in the 5α-reductase type 1 enzyme (5αRKO mice) were examined. The hypothesis was that if some of the estrous cycle differences in exploratory (open field) and anxiety (elevated plus maze) are due to P's 5α-reduction to 5α-pregnan-3α-ol-20-one (3α,5α-THP), then wildtype mice will have estrous cycle differences in the expression of these behaviors, but 5αRKO mice will not. Mice were tested in these tasks and then had plasma and brains collected so that steroid levels (estradiol, P, 3α,5α-THP, corticosterone) could be measured in these tissues. Results supported this hypothesis. There were estrous cycle differences among wildtype, but not 5αRKO, mice. Proestrous wildtype mice made more central entries in the open field and spent more time on the open arms of the plus maze, coincident with higher 3α,5α-THP levels in plasma and brain regions important for these behaviors, such as the hippocampus and cortex, compared to their diestrous counterparts. Variability in the open field and elevated plus maze could be explained by circulating and hippocampus levels of 3α,5α-THP, respectively. Thus, 5α-reductase may be required for the estrous cycle variations in affective behavior and 3α,5α-THP levels of female mice.

Mnemonic effects of progesterone to mice require formation of 3alpha,5alpha-THP.

Ovarian hormones organize and activate neural circuits for reproduction and may also mediate cognition. Research has focused on estradiol's mnemonic effects, albeit progesterone covaries with estradiol and its mechanisms for cognition require attention. Studies tested the hypothesis that cognitive effects of progesterone occur subsequent to its metabolism to 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP), which does not bind progestin receptors. Cognitive performance and progestogen levels in plasma, hippocampus, and cortex were determined in ovariectomized mice administered vehicle, or progestins that differentially form 3alpha,5alpha-THP and bind progestin receptors (progesterone, 3alpha,5alpha-THP, and/or medroxyprogesterone acetate). Only treatments that increased 3alpha,5alpha-THP levels during memory consolidation (progesterone, 3alpha,5alpha-THP, 3alpha-5alpha-THP plus medroxyprogesterone acetate, but not progesterone plus medroxyprogesterone acetate) improved cognitive performance. Thus, formation of 3alpha,5alpha-THP may be required for progesterone's cognitive-enhancing effects.

Trilostane exerts antidepressive effects among wild-type, but not estrogen receptor [beta] knockout mice.

Women with estrogen receptor (ER) positive breast cancer, who are treated with the ER blocker, tamoxifen, have an increased risk of depression. Trilostane, a 3b-hydroxysteroid dehydrogenase inhibitor, is now being used to treat tamoxifen-insensitive breast cancer. In-vitro assays show that trilostane may have actions through ERb. Results of in-vivo research shows that actions at ERb may underline some antidepressant effects of estrogen. We hypothesized that trilostane may exert antidepressive effects in the forced swim in part due to actions through ERb. Trilostane (25 mg/kg, intraperitoneally), compared with vehicle, had significant antidepressant-like effects but only when administered to wild-type, not ERb knockout, mice. Thus, actions of trilostane through ERb may underlie some of its antidepressant-like effects.