Neurosteroidogenesis Today: Novel Targets for Neuroactive Steroid Synthesis and Action and Their Relevance for Translational Research.

Neuroactive steroids are endogenous neuromodulators synthesised in the brain that rapidly alter neuronal excitability by binding to membrane receptors, in addition to the regulation of gene expression via intracellular steroid receptors. Neuroactive steroids induce potent anxiolytic, antidepressant, anticonvulsant, sedative, analgesic and amnesic effects, mainly through interaction with the GABAA receptor. They also exert neuroprotective, neurotrophic and antiapoptotic effects in several animal models of neurodegenerative diseases. Neuroactive steroids regulate many physiological functions, such as the stress response, puberty, the ovarian cycle, pregnancy and reward. Their levels are altered in several neuropsychiatric and neurological diseases and both preclinical and clinical studies emphasise a therapeutic potential of neuroactive steroids for these diseases, whereby symptomatology ameliorates upon restoration of neuroactive steroid concentrations. However, direct administration of neuroactive steroids has several challenges, including pharmacokinetics, low bioavailability, addiction potential, safety and tolerability, which limit its therapeutic use. Therefore, modulation of neurosteroidogenesis to restore the altered endogenous neuroactive steroid tone may represent a better therapeutic approach. This review summarises recent approaches that target the neuroactive steroid biosynthetic pathway at different levels aiming to promote neurosteroidogenesis. These include modulation of neurosteroidogenesis through ligands of the translocator protein 18 kDa and the pregnane xenobiotic receptor, as well as targeting of specific neurosteroidogenic enzymes such as 17ß-hydroxysteroid dehydrogenase type 10 or P450 side chain cleavage. Enhanced neurosteroidogenesis through these targets may be beneficial not only for neurodegenerative diseases, such as Alzheimer's disease and age-related dementia, but also for neuropsychiatric diseases, including alcohol use disorders.

Involvement of pregnane xenobiotic receptor in mating-induced allopregnanolone formation in the midbrain and hippocampus and brain-derived neurotrophic factor in the hippocampus among female rats.

RATIONALE: Given that the pregnane neurosteroid, 5α-pregnan-3α-ol-20-one (3α,5α-THP), is increased following behavioral challenges (e.g., mating), and that there is behavioral-induced biosynthesis of 3α,5α-THP in midbrain and mesocorticolimbic structures, 3α,5α-THP likely has a role in homeostasis and motivated reproduction and reproduction-related behaviors (e.g., affect, affiliation). The role of pregnane xenobiotic receptor (PXR), involved in cholesterol metabolism, for these effects is of continued interest. OBJECTIVES: We hypothesized that there would be differences in brain levels of 3α,5α-THP following varied behavioral experiences, an effect abrogated by knockdown of PXR in the midbrain. METHODS: Proestrous rats were infused with PXR antisense oligonucleotides (AS-ODNs) or vehicle to the ventral tegmental area before different behavioral manipulations and assessments. Endpoints were expression levels of PXR in the midbrain, 3α,5α-THP, and ovarian steroids (estradiol, progesterone, dihydroprogesterone) in the midbrain, striatum, hippocampus, hypothalamus, prefrontal cortex, and plasma. RESULTS: Across experiments, knocking down PXR reduced PXR expression and 3α,5α-THP levels in the midbrain and hippocampus. There were differences in terms of the behavioral manipulations, such that paced mating had the most robust effects to increase 3α,5α-THP levels and reduce open field exploration and social interaction. An additional question that was addressed is whether brain-derived neurotrophic factor (BDNF) is a downstream factor for regulating effects of behavioral-induced 3α,5α-THP biosynthesis. Rats infused with PXR AS-ODNs had lower levels of BDNF in the hippocampus. CONCLUSION: Thus, PXR may be a regulator of mating-induced 3α,5α-THP formation and behavioral changes and neural plasticity, such as BDNF.

Role of pregnane xenobiotic receptor in the midbrain ventral tegmental area for estradiol- and 3α,5α-THP-facilitated lordosis of female rats.

RATIONALE: Progesterone and its metabolite, 5α-pregnan-3α-ol-20-one (3α,5α-THP), have actions in the ventral tegmental area (VTA) that are required for lordosis, a characteristic mating posture of female rodents. 17ß-estradiol (estradiol) co-varies with progestogens over natural cycles, enhances production of 3α,5α-THP, and is required for successful reproductive behavior. OBJECTIVES: A question of interest is the role of pregnane xenobiotic receptor (PXR), a nuclear receptor that regulates enzymes needed for the production of 3α,5α-THP, for estradiol-mediated lordosis. The hypothesis tested was that if PXR is involved in estradiol-mediated biosynthesis of 3α,5α-THP and reproductive behavior, knocking down expression of PXR in the VTA of estradiol-primed, but not vehicle-primed, rats should decrease lordosis and midbrain 3α,5α-THP; effects may be attenuated by 3α,5α-THP administered to the VTA. METHODS: Ovariectomized rats were administered subcutaneous injections of oil vehicle or estradiol. Rats were then administered PXR antisense oligonucleotides (PXR AS-ODNs; which are expected to locally knock down expression of PXR), or control (saline), infusions to the VTA. Rats were administered 3α,5α-THP or vehicle via infusions to the VTA. Reproductive behavior (paced mating task) of rats was determined in addition to exploratory (open field), affective (elevated plus maze), and pro-social (social interaction task) behavior. RESULTS: Reproductive behavior (i.e., increased lordosis) was enhanced with estradiol-priming and infusions of 3α,5α-THP to the VTA. Infusions of PXR AS-ODNs to the VTA attenuated responses in estradiol-, but not vehicle-, primed rats, compared to control infusions. CONCLUSIONS: PXR may be involved in a neuroregulatory response involving biosynthesis of 3α,5α-THP in the midbrain VTA of estradiol-primed rats.

Pregnane xenobiotic receptors and membrane progestin receptors: role in neurosteroid-mediated motivated behaviours.

Progestogens have actions in the midbrain ventral tegmental area (VTA) to mediate motivated behaviours, such as those involved in reproductive processes, among female rodents. In the VTA, the formation and actions of one progestogen, 5α-pregnan-3α-ol-20-one (3α,5α-THP), are necessary and sufficient to facilitate sexual responding (measured by lordosis) of female rodents. Although 3α,5α-THP can be produced after metabolism of ovarian progesterone, 3α,5α-THP is also a neurosteroid produced de novo in brain regions, such as the VTA. There can be dynamic changes in 3α,5α-THP production associated with behavioural experience, such as mating. Questions of interest are the sources and targets of 3α,5α-THP. Regarding sources, the pregnane xenobiotic receptor (PXR) may be a novel factor involved in 3α,5α-THP metabolism in the VTA (as well as a direct target of 3α,5α-THP). We have identified PXR in the midbrain of female rats, and manipulating PXR in this region reduces 3α,5α-THP synthesis and alters lordosis, as well as affective and social behaviours. Regarding targets, recent studies have focused on the role of membrane progestin receptors (mPRs). We have analysed the expression of two of the common forms of these receptors (mPRα/paqr7 and mPRß/paqr8) in female rats. The expression of mPRα was observed in peripheral tissues and brain areas, including the hypothalamus and midbrain. The expression of mPRß was only observed in brain tissues and was abundant in the midbrain and hypothalamus. To our knowledge, studies of these receptors in mammalian models have been limited to expression and regulation, instead of function. One question that was addressed was the functional effects of progestogens via mPRα and mPRß in the midbrain of hormone-primed rats for lordosis. Studies to date suggest that mPRß may be an important target of progestogens in the VTA for lordosis. Taken together, the result of these studies demonstrate that PXR is involved in the production of 3α,5α-THP in the midbrain VTA. Moreover, mPRs may be a target for the actions of progestogens in the VTA for lordosis.

Milestones on Steroids and the Nervous System: 10 years of basic and translational research.

During the last 10 years, the conference on 'Steroids and Nervous System' held in Torino (Italy) has been an important international point of discussion for scientists involved in this exciting and expanding research field. The present review aims to recapitulate the main topics that have been presented through the various meetings. Two broad areas have been explored: the impact of gonadal hormones on brain circuits and behaviour, as well as the mechanism of action of neuroactive steroids. Relationships among steroids, brain and behaviour, the sexual differentiation of the brain and the impact of gonadal hormones, the interactions of exogenous steroidal molecules (endocrine disrupters) with neural circuits and behaviour, and how gonadal steroids modulate the behaviour of gonadotrophin-releasing hormone neurones, have been the topics of several lectures and symposia during this series of meetings. At the same time, many contributions have been dedicated to the biosynthetic pathways, the physiopathological relevance of neurosteroids, the demonstration of the cellular localisation of different enzymes involved in neurosteroidogenesis, the mechanisms by which steroids may exert some of their effects, both the classical and nonclassical actions of different steroids, the role of neuroactive steroids on neurodegeneration, neuroprotection, and the response of the neural tissue to injury. In these 10 years, this field has significantly advanced and neuroactive steroids have emerged as new potential therapeutic tools to counteract neurodegenerative events.

Endocrine disrupters: a review of some sources, effects, and mechanisms of actions on behaviour and neuroendocrine systems.

Some environmental contaminants interact with hormones and may exert adverse consequences as a result of their actions as endocrine disrupting chemicals (EDCs). Exposure in people is typically a result of contamination of the food chain, inhalation of contaminated house dust or occupational exposure. EDCs include pesticides and herbicides (such as dichlorodiphenyl trichloroethane or its metabolites), methoxychlor, biocides, heat stabilisers and chemical catalysts (such as tributyltin), plastic contaminants (e.g. bisphenol A), pharmaceuticals (i.e. diethylstilbestrol; 17α-ethinylestradiol) or dietary components (such as phytoestrogens). The goal of this review is to address the sources, effects and actions of EDCs, with an emphasis on topics discussed at the International Congress on Steroids and the Nervous System. EDCs may alter reproductively-relevant or nonreproductive, sexually-dimorphic behaviours. In addition, EDCs may have significant effects on neurodevelopmental processes, influencing the morphology of sexually-dimorphic cerebral circuits. Exposure to EDCs is more dangerous if it occurs during specific 'critical periods' of life, such as intrauterine, perinatal, juvenile or puberty periods, when organisms are more sensitive to hormonal disruption, compared to other periods. However, exposure to EDCs in adulthood can also alter physiology. Several EDCs are xenoestrogens, which can alter serum lipid concentrations or metabolism enzymes that are necessary for converting cholesterol to steroid hormones. This can ultimately alter the production of oestradiol and/or other steroids. Finally, many EDCs may have actions via (or independent of) classic actions at cognate steroid receptors. EDCs may have effects through numerous other substrates, such as the aryl hydrocarbon receptor, the peroxisome proliferator-activated receptor and the retinoid X receptor, signal transduction pathways, calcium influx and/or neurotransmitter receptors. Thus, EDCs, from varied sources, may have organisational effects during development and/or activational effects in adulthood that influence sexually-dimorphic, reproductively-relevant processes or other functions, by mimicking, antagonising or altering steroidal actions.

Inhibition of 5α-reductase activity in late pregnancy decreases gestational length and fecundity and impairs object memory and central progestogen milieu of juvenile rat offspring.

Psychological, physical and/or immune stressors during pregnancy are associated with negative birth outcomes, such as preterm birth and developmental abnormalities. In rodents, prenatal stressors can alter the expression of 5α-reductase enzymes in the brain and may influence cognitive function and anxiety-type behaviour in the offspring. Progesterone plays a critical role in maintaining gestation. In the present study, it was hypothesised that 5α-reduced progesterone metabolites influence birth outcomes and/or the cognitive and neuroendocrine function of the offspring. 5α-Reduced steroids were manipulated in pregnant Long-Evans rats via the administration of vehicle, the 5α-reduced, neuroactive metabolite of progesterone, 5α-pregnan-3α-ol-20-one (3α,5α-THP, allopregnanolone; 10 mg/kg/ml, s.c.), or the 5α-reductase inhibitor, finasteride (50 mg/kg/ml, s.c.), daily from gestational days 17-21. Compared to vehicle or 3α,5α-THP treatment, finasteride, significantly reduced the length of gestation and the number of pups per litter found in the dams' nests after parturition. The behaviour of the offspring in hippocampus-dependent tasks (i.e. object recognition, open field) was examined on post-natal days 28-30. Compared to vehicle-exposed controls, prenatal 3α,5α-THP treatment significantly increased motor behaviour in females compared to males, decreased progesterone content in the medial prefrontal cortex (mPFC) and diencephalon, increased 3α,5α-THP and 17ß-oestradiol content in the hippocampus, mPFC and diencephalon, and significantly increased serum corticosterone concentrations in males and females. Prenatal finasteride treatment significantly reduced object recognition, decreased hippocampal 3α,5α-THP content, increased progesterone concentration in the mPFC and diencephalon, and increased serum corticosterone concentration in female (but not male) juvenile offspring, compared to vehicle-exposed controls. Thus, inhibiting the formation of 5α-reduced steroids during late gestation in rats reduces gestational length, the number of viable pups per litter, and impairs cognitive and neuroendocrine function in the juvenile offspring.

Progesterone turnover to its 5α-reduced metabolites in the ventral tegmental area of the midbrain is essential for initiating social and affective behavior and progesterone metabolism in female rats.

BACKGROUND: Among women and female rodents, progesterone (P) influences social affiliation and affect. These effects may be partly due to formation of its 5α-reduced, 3α- hydroxylated metabolite, 5α-pregnan-3α-ol-20-one (3α,5α- THP). AIM: To elucidate whether actions of 3α,5α-THP in the midbrain ventral tegmental area (VTA) are both necessary and sufficient to enhance non-sexual and sexual social behaviors, affect, and central 3α,5α-THP metabolism. MATERIALS AND METHODS: P and 3α,5α-THP formation were unperturbed or blocked in VTA via infusions of vehicle, PK11195 (400 ng), and/or indomethacin (10 µg). Rats then received subsequent infusions of vehicle or 3α,5α-THP (100 ng) and were assessed in a battery of tasks that included open field (exploration), elevated plus maze (anxiety behavior), social interaction (social affiliation), and paced mating (sexual behavior) or were not tested. Metabolic turnover of P to its 5α-reduced metabolites was assessed in plasma, midbrain, hippocampus, frontal cortex, diencephalon, and remaining subcortical tissues (control interbrain). RESULTS: Infusions of any combination of inhibitors significantly reduced social and affective behavior in all tasks compared to vehicle, concomitant with reduced turnover of P to its 5α-reduced metabolites, in midbrain only. Subsequent infusions of 3α,5α-THP significantly reinstated/enhanced anti- anxiety behavior, lordosis, and P turnover to its 5α-reduced metabolites in midbrain, as well as hippocampus, cortex, and diencephalon (but not plasma or interbrain). CONCLUSIONS: These data are the first to provide direct evidence that actions of 3α,5α-THP in the VTA are both necessary and sufficient for social and affective behavior, as well as initiation of central 5α-reduction.

Sex-dependent effects of chronic unpredictable stress in the water maze.

Exposure to chronic predictable stress, such as restraint, can affect performance on spatial memory tasks and these effects have been shown to be sex-specific in rats. It is not known whether unpredictable stress has similar sex-specific effects on spatial memory and whether those effects are present after the stress procedure has ended. Therefore, the current study tested male and female rats in the Morris water maze either immediately or 3 weeks following exposure to 10 days of unpredictable stress (CUS). Male and female rats were exposed to 10 days of stressors that varied by type and time of stressor application. Exposure to CUS decreased the distance swam to locate the hidden platform during acquisition training in the water maze for female but not male rats. Overall, male rats performed better than female rats during the acquisition, probe and matching to place trials. These effects were observed when assessing spatial memory performance immediately or 3 weeks following the last stressor. Plasma corticosterone levels followed the behavioral differences during the acquisition trials in that control female rats had increased basal and swim-stimulated corticosterone levels compared to CUS female rats and control male rats. These data demonstrate that unpredictable stress influences performance on the water maze in a sex-specific manner, which parallel plasma corticosterone levels. The improved performance of female rats following CUS exposure was present 3 weeks after the termination of the stress procedures, suggesting that stress may have lasting effects on underlying neural systems.

Male gamblers have significantly greater salivary cortisol before and after betting on a horse race, than do female gamblers.

Prevalence rates of gambling are influenced by gender. Among normative populations, hypothalamic-pituitary-adrenal (HPA) axis response to stress is affected by gender. However, pathological, compared to recreational, gamblers demonstrate perturbations in HPA activation in response to gambling stimuli. We examined whether there were gender differences in HPA response to gambling in a naturalistic setting among horse-race bettors and scratch-off lottery bettors. Salivary cortisol was collected from horse-race gamblers (n=32) and scratch-off lottery ticket players (n=39) before and after (0, 10, or 20 min) betting on a horse race at an off-track betting establishment. Salivary cortisol levels were significantly higher among men than among women, both prior to and following, betting on a horse race. Among women, but not men, there was a decline in salivary cortisol across time in scratch-off bettors, whereas women horse-race bettors maintained consistent low concentrations of salivary cortisol at every time point sampled. Together these data suggest that engaging in gambling may have different effects on stress responses of men, compared to women. Whether these gender differences in HPA activation contribute to gender-related differences in gambling behavior is the subject of ongoing investigation.

Gambling pathology is associated with dampened cortisol response among men and women.

Pathological gambling has many similarities to pharmacological addiction. Notably, both pathological gambling and drug addiction are characterized by aberrations in hypothalamic-pituitary-adrenal (HPA) axis responding. As well, there are indications that gender differences may play a role in these processes. Whether gender and/or HPA response are associated with pathological gambling was of interest. Recreational and pathological gamblers (15 men and 6 women per group) had the HPA factor, cortisol, assessed in saliva before and after watching a video of their preferred mode of gambling (slot machines, horse race betting, scratch-off tickets, blackjack, video poker, craps, sports betting, online casino games, or lottery tickets), and a video of neutral stimuli (a rollercoaster ride). Basal levels of salivary cortisol did not significantly differ among recreational and pathological gamblers. However, recreational gamblers demonstrated significantly increased salivary cortisol levels after the gambling and rollercoaster videos, whereas pathological gamblers demonstrated no salivary cortisol increase in response to either video stimulus. There was also a non-significant trend for women to have a greater cortisol response to video stimuli compared to men. These data suggest that pathological gambling is associated with hypoactive HPA response to gambling stimuli, similar to chronic drug exposure, and gender may contribute to this effect.

Steroids, reproductive endocrine function, and affect. A review.

Although the effects of estrogen (E2) on mood have been studied for some time, there is controversy over the utility of hormone replacement therapy (HRT). Administration of E2 and/or other steroid hormones (e.g., progestogens, androgens, etc.) may be able to reduce increased anxiety and depression that is present with the onset of menopause. However, some studies indicate that E2 replacement does not significantly improve anxiety and/or depressive symptoms in all postmenopausal subjects. More recent data suggests that the efficacy of HRT could be based on a number of factors, including variety of E2-replacements available, the timing during or after menopause when HRT is initiated, and/or effects of other steroid hormones, such as progestogens and androgens. Notably, little attention has been paid to the possible synergistic effects of E2 that may require progestogens and/or androgens to produce positive outcomes in mood. Additionally, steroid hormones have a number of effects that influence anxiety and depression across the lifespan. As such, dose, timing, and combination of steroid replacement may explain these differences in behavioral outcome. With the increasing peri- to postmenopausal population, many women can expect to live nearly half their lifetime in a postmenopausal state. Therefore, examining these ambiguous findings is of critical importance. This review will focus on a synthesis of the available information regarding findings from animal and human studies in terms of effects of steroid hormones across the lifespan, different HRT options and their subsequent interactions in the brain and/or the hypothalamic-pituitary-adrenal axis, and effects on anxiety and depression.

Steroids, reproductive endocrine function, and cognition. A review.

Findings regarding post-menopausal effects of estrogen (E2) on women's cognition are divergent. Observational studies of post-menopausal women and a large body of basic research indicates E2 is able to reduce cognitive decline. For instance, the Women's Health Initiative Study, which examined women 64 years or older who were randomly assigned to receive hormone-replacement therapy (HRT), found a two-fold increase in dementia diagnoses among this population. Since then, several theories have emerged to explain such discrepant findings, such as the variety of E2-replacements available, the timing after menopause when HRT is initiated, and/or the effects of other steroid hormones (e.g. progestins, androgens, etc). In fact, considerably less focus has been paid to the potential synergistic input E2 may require from progestins and/or androgens in mediating these processes. Due to the increasing peri- to post-menopausal population, and the ambiguous findings from bench to clinical trials, this review will focus on a synthesis of the available information regarding findings from animal and human studies in terms of different HRT options and their subsequent interactions in the brain, effects on womens' cognitive abilities and risk of developing a neurodegenerative disease.

Adult female wildtype, but not oestrogen receptor beta knockout, mice have decreased depression-like behaviour during pro-oestrus and following administration of oestradiol or diarylpropionitrile.

Studies in people and animal models suggest that depression is influenced by natural fluctuations in the levels of 17beta-oestradiol (E(2)), as well as administration of E(2)-based therapies, such as selective oestrogen receptor modulators (SERMs). Elucidating the effects and mechanisms of E(2) is important to improve future E(2)-based therapeutics. An important question is whether effects of E(2) or SERMs for mood regulation act at the alpha or beta isoform of the oestrogen receptor (ER) because some of the unwanted trophic effects of E(2)-based therapies may involve actions at ERalpha, rather than ERbeta. In the present study, whether there are sex differences in depression-like behaviour of adult mice (experiment 1), and the effects of natural fluctuations in E(2) (experiment 2), or administration of E(2) or a SERM that has higher affinity for ERbeta than for ERalpha (diarylpropionitrile; DPN) to ovariectomised (experiment 3) wildtype and ERbeta knockout (betaERKO) mice were investigated. Results of this study supported our hypotheses that: there would be sex differences favouring males for depression-like behaviour and endogenous increases in, or exogenous administration of, E(2) or administration of an ERbeta SERM would decrease depression-like behaviour in wildtype, but not betaERKO, mice. In experiment 1, adult male mice spent less time immobile in the forced swim test (i.e., showed less depression-like behaviour) compared with female mice. In experiment 2, pro-oestrous (higher circulating E(2) levels), compared with dioestrous (lower circulating E(2) levels), mice had reduced immobility in the forced swim test; this effect was not observed in betaERKO mice. In experiment 3, administration of E(2) or DPN to ovariectomised wildtype, but not betaERKO, mice decreased immobility compared with vehicle administration, these data suggest that ERbeta may be required for some of the anti-depressant-like effects of E(2).

Estrogen is necessary for 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP) infusion to the ventral tegmental area to facilitate social and sexual, but neither exploratory nor affective behavior of ovariectomized rats.

The progesterone metabolite, 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP, allopregnanolone), acts in the ventral tegmental area (VTA) to facilitate exploratory, anti-anxiety, and socio-sexual behavior among ovariectomized (OVX), estrogen (E(2))-primed rats and gonadally-intact rats with high (proestrus) or low (diestrus) endogenous E(2) levels. The extent to which E(2) is required for these effects of 3alpha,5alpha-THP is not known. OVX rats were primed with systemic 17beta-estradiol (10 microg) or oil vehicle and were infused 44 h later with 3alpha,5alpha-THP (100 ng) or beta-cyclodextrin vehicle to the VTA, substantia nigra (SN), or central grey (CG). Rats were assessed in a battery of exploratory (open field), anxiety (elevated plus maze), social (partner preference, social interaction), and sexual (paced mating) tasks. E(2)-priming was necessary for 3alpha,5alpha-THP infusions to facilitate social interaction and mating and midbrain 3alpha,5alpha-THP levels were higher among E(2)-compared to vehicle-primed rats. Irrespective of E(2)-priming, rats infused with 3alpha,5alpha-THP to the VTA, but not SN or CG, demonstrated increased exploration in an open field, anti-anxiety behavior on an elevated plus maze, and preference for a male. Thus, actions of 3alpha,5alpha-THP in the VTA to enhance social and sexual behaviors were reliant on E(2) but increases in exploratory and anti-anxiety behavior were not.

In the ventral tegmental area, the membrane-mediated actions of progestins for lordosis of hormone-primed hamsters involve phospholipase C and protein kinase C.

Progestin-facilitated lordosis of rodents is enhanced by activation of dopamine type 1 (D(1)) or GABA(A) receptors, their downstream G-proteins, and/or second messengers in the ventral tegmental area (VTA). We examined whether the ability of progestins to enhance lordosis via actions at D(1) and/or GABA(A) receptors is contingent upon activation of the second messenger phospholipase C (PLC) and its associated kinase, protein kinase C (PKC), in the VTA. If the actions of progestins through D(1) and GABA(A) receptors in the VTA are mediated through PLC and PKC, then inhibiting PLC formation (Experiment 1) or blocking PKC (Experiment 2) should reduce progestin-facilitated lordosis and its enhancement by D(1) (SKF38393) or GABA(A) (muscimol) receptor agonists. In Experiment 1, ovariectomised hamsters, primed with oestradiol (10 microg; h 0) + progesterone (100 microg; h 45), were pretested for lordosis and motor behaviour (h 48) and then infused with the PLC inhibitor, U73122 (400 nM/side), or vehicle. Thirty minutes later, hamsters were retested and then received infusions of SKF38393 (100 ng/side), muscimol (100 ng/side), or vehicle to the VTA. Hamsters were post-tested for lordosis and motor behaviour 30 min later. In Experiment 2, a similar protocol was utilised except that instead of the PLC inhibitor hamsters were infused with the PKC inhibitor, bisindolylmaleimide (75 nM/side). Systemic progesterone, SKF38393-, and muscimol-facilitated lordosis was attenuated by infusion of the PLC inhibitor, U73122, or the PKC inhibitor, bisindolylmaleimide, compared to vehicle to the VTA. Thus, the actions of progestins in the VTA to enhance lordosis through D(1) and/or GABA(A) may include downstream activity of PLC and PKC.

In the ventral tegmental area, cyclic AMP mediates the actions of progesterone at dopamine type 1 receptors for lordosis of rats and hamsters.

Progesterone-facilitated lordosis is enhanced by activation of, and inhibited by antagonism of, dopamine type 1 receptors (D1) in the ventral tegmental area (VTA). Given that D1 activation leads to increases in cyclic AMP (cAMP), we hypothesised that, in the VTA, progesterone's actions on lordosis that involve D1 are mediated, in part, by cAMP. In Experiment 1, naturally receptive rats and hamsters were pretested for lordosis, infused with the cAMP analogue 8-bromo-cAMP (200 ng) or vehicle to the VTA, and tested again 30 min later. In Experiments 2 and 3, ovariectomised, oestradiol (10 microg) + progesterone (0 or 100 microg)-primed rats and oestradiol (10 microg) + progesterone (0 or 200 microg)-primed hamsters were pretested for lordosis and infused with 8-bromo-cAMP (200 ng), the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine (12 microM) or vehicle to the VTA. Subjects were tested again 30 min later. In Experiment 4, oestradiol + progesterone-primed rats and hamsters were pretested and infused with the D1 agonist SKF38393 (0 or 100 ng) to the VTA. Thirty minutes later, subjects were tested again and infused with 2',5'-dideoxyadenosine (12 microM) or vehicle. Subjects were tested again 30 min later. VTA infusions of 8-bromo-cAMP enhanced lordosis of naturally receptive or hormone-primed rats and hamsters and 2',5'-dideoxyadenosine decreased lordosis of oestradiol + progesterone-primed rats and hamsters. D1-mediated increases in progesterone-facilitated lordosis were reduced by 2',5'-dideoxyadenosine. These data suggest that progesterone-facilitated lordosis of rats and hamsters may be modulated by D1 and cAMP activity in the VTA.

Infusions of 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP) to the ventral tegmental area, but not the substantia nigra, enhance exploratory, anti-anxiety, social and sexual behaviours and concomitantly increase 3alpha,5alpha-THP concentrations in the hippocampus, diencephalon and cortex of ovariectomised oestrogen-primed rats.

Sequential actions of 17beta-oestradiol (E2) and progesterone (P4) in the ventromedial hypothalamus (VMH) and ventral tegmental area (VTA) mediate sexual behaviour of female rodents. In the presence of appropriate environmental stimuli, E2 and P4 can facilitate initiation of sex behaviour (i.e. lordosis), in part through classic actions at intracellular progestin receptors in the VMH. However, the effects of P4 in the VTA to modulate lordosis involve its metabolite, 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP), which can have paracrine effects in the brain to reduce anxiety and stress. We investigated the effects of 3alpha,5alpha-THP infusions to the VTA, and a control site, the substantia nigra (SN), on exploratory, anti-anxiety, social and sexual behaviours (socio-sexual behaviours) and hormone levels in the midbrain and other regions (hippocampus, diencephalon and cortex) that may mediate these functions. Ovariectomised, rats were E2-primed (10 microg, s.c.) at 0 h and were infused with beta-cyclodextrin vehicle or 3alpha,5alpha-THP to the VTA or SN 44-48 h later. Ten minutes after infusions, rats were tested in the open field, plus maze, partner preference, social interaction and paced mating tasks, or served as nontested controls. Infusions of 3alpha,5alpha-THP to the VTA, but not the SN, increased central entries in the open field, open arm time in the plus maze, time spent in proximity to a male, duration of social interaction, incidence and intensity of lordosis, pacing, proceptivity, and anti-conflict behaviour. 3Alpha,5alpha-THP, but not vehicle, infusions to the VTA (but not the SN) also increased 3alpha,5alpha-THP levels in the midbrain, as well as the hippocampus, diencephalon and cortex. Behavioural testing increased levels of the precursor of 3alpha,5alpha-THP precursor, dihydroprogesterone (DHP). Thus, infusions of 3alpha,5alpha-THP to the VTA enhance socio-sexual behaviours and increase 3alpha,5alpha-THP levels in the hippocampus, diencephalon and cortex, and behavioural testing increases DHP levels in brain areas involved in modulating socio-sexual behaviours.

Rats bred for high versus low anxiety responses neonatally demonstrate increases in lordosis, pacing behavior, and midbrain 3 alpha, 5 alpha-THP levels as adults.

Duration and intensity of lordosis is mediated by actions of the progesterone (P) metabolite, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-THP) at GABA-sub(A) receptors in the midbrain ventral tegmental area. Because rats selectively bred for infantile vocalizations may differ in endogenous 3 alpha,5 alpha-THP secretion, their sexual behavior, midbrain, and plasma 3 alpha,5 alpha-THP levels as adult rats in behavioral estrus was examined. Rats bred for high rates of infantile vocalizations had shorter latencies and intervals between intromissions and ejaculation, higher lordosis quotients and ratings, more pacing of their sexual contacts, and had higher P and 3 alpha,5 alpha-THP levels in plasma and midbrain than did rats bred for low rats of infantile vocalizations. Thus, levels of 3 alpha,5 alpha-THP in the midbrain are associated with differences in sexual behavior of these rats.

Progesterone's effects to reduce anxiety behavior of aged mice do not require actions via intracellular progestin receptors.

RATIONALE: Aging is associated with reduced secretion of, and down-regulation of receptors for, progesterone (P); yet, P's effects when administered to younger and older animals have not been systematically investigated. Some of P's antianxiety effects may be due to its conversion to 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) and its subsequent actions as a positive modulator at GABAA receptor complexes (GBRs). OBJECTIVES: We investigated whether P administration can decrease anxiety behavior of progestin receptor (PR) knockout (PRKO) or wild-type control mice. METHODS: P (10 mg/kg) or vehicle (propylene glycol) were administered subcutaneously to intact, female or male wild-type or PRKO mice that were either 9-12 or 18-24 months of age. Behavior in tasks that assess spontaneous activity (activity monitor and roto-rod), free exploration of a novel environment (open field, elevated plus maze, and elevated zero maze), and conflict behavior (mirror chamber, dark-light transition, and punished drinking) were examined 1 h after injection. RESULTS: P significantly decreased anxiety behavior of both PRKO and wild-type mice. P did not alter motor behavior but increased central entries in the open field, time in the open quadrants of the elevated zero maze, time in the mirrored chamber, time in the light compartment of the dark-light transition, and punished drinking in young and old mice. P-administered mice had higher levels of hippocampal 3alpha,5alpha-THP and GABA-stimulated chloride flux than did vehicle-administered PRKO or wild-type mice. CONCLUSIONS: The effects of P to decrease anxiety behavior of younger and older mice do not require classic PRs and may involve actions of 3alpha,5alpha-THP at GBRs.