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

Abstract

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.

INTRODUCTION

Investigations aiming to elucidate the dynamic relationship between social interactions and neurobiological response have traditionally been understudied; however, this has begun to shift in recent years (1). In particular, the steroid hormone, progesterone (P), which is secreted from peripheral glands (ovaries and adrenals) of women and female rodents, cyclically and in response to stress, may play an important and dynamic role in social affiliation. Among women, there are small, but consistent, effects for increases in P around the ovulatory phase of the menstrual cycle, to influence preference for others’ physical features (2-8) and motivation to affiliate (9,10). These effects may be dynamic, given that social contact and enhancement of social motivation are associated with elevated circulatory P in women (11-13). In animal models, similar effects are observed wherein social affiliation is enhanced when P is cyclically-increased (14). Extirpation of peripheral P decreases pro-social behavior (15) and systemic P replacement can attenuate anti-social effects of agonistic hormone-priming regimen (16,17). However, the mechanisms that underlie P's effects to influence social responding are understudied and have not been well-characterized.

Engagement in social networking may also be an important factor underlying positive mental health (18,19) and P is an important modulator of affective behavior in people and animals. Among women, anxiety and stress are reduced when endogenous progestogens are elevated cyclically or during pregnancy (20-23). Conversely, the risk for development of neuropsychiatric symptoms is greatest when endogenous P is reduced premenstrually, post-partum, or post-menopausally (21,24-30). Fluctuations in endogenous P may partly underlie these effects given that they can be ameliorated by progestogen administration (31-33). These findings are recapitulated in animal models (14,34-40) and may partly occur via P's metabolism to its 5α-reduced metabolite, dihydroprogesterone (DHP), followed by 3α-hydroxylation to, 5α-pregnan-3α-ol-20-one (3α,5α-THP; a.k.a. allopregnanolone). When P's conversion to its 5α-reduced metabolites are blocked, anxiety-like behavior is increased (41,42). Direct infusion of 3α,5α-THP, or infusion of a 3α,5α-THP enhancer, to dorsal hippocampus has been reported to reduce anxiety behavior in rats (43,44), albeit, this is not always observed (45). As such, P may have anti-anxiety effects, in part, via its metabolism to 3α,5α-THP in hippocampus; however, these actions do not account for all of P's anti-anxiety effects.

Social experiences occur rapidly and normative behavioral response involves many brain regions, including hippocampus, but also cortical and midbrain regions (46-49). P can be formed de novo in brain from glial and neuronal cells, to rapidly alter social behavior. In rodent models, P's cyclical enhancements coincide with the onset of sexual-receptivity (operationally-defined as lordosis). We and others have utilized expression of lordosis as a bioassay to assess P's underlying mechanisms (50-55). In particular, these investigations have revealed de novo production of 3α,5α-THP in the midbrain ventral tegmental area (VTA) to play an important role in maintaining lordosis and enhancing sexual proceptivity (14,56). In support, infusions of 3α,5α-THP to the VTA or, enhancement of neurosteroidogenesis in this region, increases expression of lordosis and this is not abolished by ovarian or adrenal extirpation among estradiol (E)-primed rodents, suggesting that these effects may be independent of peripheral progestogen formation (14,56). Notably, these effects appear to be dynamically-modulated by the social environment, given that engagement in a sexual social behavior, mating, is associated with enhancement of 3α,5α-THP formation in midbrain and related brain regions including hippocampus, cortex, and diencephalon (14). As such, central formation of 3α,5α-THP in the midbrain VTA is an important mediator of sexual social behavior in rodents. Given that enhancement of 3α,5α-THP in VTA is associated with 3α,5α-THP formation in other brain regions, including hippocampus, the importance of this mechanism for non-sexual social behavior and affect is of interest.

Understanding the central mechanisms that underlie P's actions to modulate sexual/non-sexual social and affective behavior of rodents is critical for therapeutic intervention among individuals with related disorders. In people and rodents, all enzymes necessary to form 3α,5α-THP from cholesterol, or its progestogen precursors, are found in the adult central nervous system (57-59). In particular, 5α-reductase is richly expressed in adult midbrain and hippocampus (60-64). Notably, circulatory 3α,5α-THP formation is perturbed in women diagnosed with depression, premenstrual syndrome, or schizophrenia (65-67). 5α-reduction may also be important in the efficacy of progestins’ social and affective modulation given that synthetic progestins that cannot be 5α-reduced, such as medroxyprogesterone acetate, do not enhance sexual or affective behavior (68,69) and pharmacologically inhibiting 5α-reductase activity reduces 3α,5α-THP and attenuates anti-anxiety, anti-depression, and sexual behavior of rodents (14,41). Indeed, the efficacy of some anti-depressant and anti-psychotic medications may be dependent on their ability to enhance 3α,5α-THP in people and rodents (28,70-72). 3α,5α-THP can dampen activation of the stress axis (73) which, in animal models, directly modulates social responding and affiliation (74). As such, metabolism to 3α,5α-THP may underlie beneficial effects of P and some clinically-used therapeutics, but the role that central 3α,5α-THP formation in the midbrain plays in these effects needs to be systematically examined.

This investigation aimed to establish the critical role that centrally-formed 3α,5α-THP may play in sexual and non-sexual social and affective behavior in the midbrain VTA. We have previously seen that inhibition of 3α,5α-THP can negatively impact such behavior and enhancement of neurosteroidogenesis can partly ameliorate these effects (56). However, whether these effects are due specifically to actions of centrally-derived 3α,5α-THP is not known. To investigate the necessity of intra-VTA 3α,5α-THP on sexual/non-sexual and affective behavior, an inhibitor of P formation from cholesterol (PK11195) and/or an inhibitor of 3α,5α-THP formation from its precursor, DHP (indomethacin), were infused to the VTA and behavior was assessed. In order to establish the sufficiency of intra-VTA 3α,5α-THP for these behaviors, physiological concentrations of intra-VTA 3α,5α-THP were reinstated in some rats. Some rats were infused but not tested in the behavioral battery to assess the role of sexual engagement on central progestogen formation. We anticipated that (1) inhibition of central formation of 3α,5α-THP in the VTA would attenuate social and affective behavior, and (2) mating-induced enhancement of social and affective behavior would be due to enhanced activity of 5α-reductase, and P turnover to its 5α-reduced metabolites would be enhanced in midbrain and/or associated regions (hippocampus, cortex, diencephalon) among mated, but not non-tested, rats. (3) We expected rats that did not engage in mating to demonstrate less conversion of P to its 5α-reduced metabolites compared to rats that did engage in mating.

MATERIALS AND METHODS

These methods were pre-approved by the Institutional Animal Care and Use Committee at The University at Albany-SUNY.

Animals and Housing

Adult, intact, Long-Evans, female rats (N=293) obtained from the breeding colony of the Life Sciences Research Laboratory Animal Care Facility at The University at Albany-SUNY (original stock Charles River, Raleigh, NC) were group-housed in a temperature- and humidity-controlled room on a reverse light cycle (lights off at 08:00 h) with free access to water and Purina rat chow in their cages.

Surgery

All rats were stereotaxically implanted with bilateral guide cannulae aimed at the medial aspect of the VTA (from bregma: AP = -5.3, ML = ±0.4, DV = -7.0; 75) under xylazine (12 mg/kg) and ketamine (60 mg/kg) anesthesia. Guide cannulae were modified 23-gauge thin-wall stainless steel needles. Following surgery, animals were monitored for loss of weight, righting response, flank stimulation response, and/or muscle tone (76). There were 8 rats that failed these assessments. These rats were killed immediately and excluded from all analyses.

Determination of Sexual Receptivity

To determine when rats were in behavioral estrus, females were vaginally-masked (so as to minimize vaginocervical stimulation that could otherwise alter central hormone milieu; see 77,78). Briefly, rats were placed in an arena with a stimulus male that had been conditioned to exhibit high, consistent levels of sexual behavior. Females that exhibited receptive (lordosis) and proceptive behaviors (hopping, darting, ear wiggling) in response to one mount were considered to be in behavioral estrus and were tested on that day. This screening was conducted daily between 10:00 and 11:00 h.

Drug Preparation

PK11195 (Alexis Biomedicals, Inc, San Diego, CA), an 18 kDa translocator protein (TSPO) antagonist, was used to attenuate biosynthesis of 3α,5α-THP in the VTA. PK11195 was prepared to a concentration of 400 ng/μl in sterile saline with 1% DMSO and 2 drops of Tween 80. Briefly, 2 mg of crystalline PK11195 were weighed out in a 1.5 ml tube and a solution of 2.5 μl DMSO and two small drops of Tween 80 were added. The solution was vortexed well and transferred to a 10 ml vial. Next, 2.5 ml saline were added in 0.5 ml increments, with vortexing occurring after each 0.5 ml increment. We have used this regimen previously to decrease anti-anxiety effects of 3α,5α-THP, when infused to hippocampus (43) and to reduce midbrain 3α,5α-THP levels when infused to VTA concomitant with lordosis and anti-anxiety behavior (14,56).

Indomethacin (Sigma Chemical Co., St Louis, MO), is an inhibitor of the 3α-hydroxysteroid dehydrogenase (3α-HSD) enzyme that catalyzes the conversion of DHP to 3α,5α-THP. Indomethacin was prepared to a concentration of 10 μg/μl in sterile saline with 25% β-cyclodextrin. We have previously found this dosing regimen to reduce 3α,5α-THP concentrations in midbrain concomitant with lordosis and anti-anxiety behavior when infused to the VTA (14,56). It is notable that indomethacin also blocks cyclooxygenase-2, suppressing prostaglandin production. Some non-specific effects of indomethacin have been reported (including ~50% reduction of locomotor and grooming behavior) when administered via jugular catheter ~6 mg/kg (79). However, indomethacin is also shown to be highly selective for 3α-HSD (80) and, when infused to the VTA, reveals a similar pattern of effects as pharmacological PK11195 and 5α-reductase inhibitors on lordosis (14,56). As such, we assessed locomotor behavior, in addition to social, affective, and sexual behavior in order to assess non-specific effects of drug regimen utilized.

3α,5α-THP (purchased from Dr. Robert Purdy, Scripps Institute, CA) was prepared to a concentration of 100 ng/μl in sterile saline. This concentration of 3α,5α-THP produces midbrain 3α,5α-THP levels in ovariectomized rodents akin to that of rats in natural, behavioral estrus (14,56).

Procedure

Rats were randomly assigned to one of eight groups, and either tested in the behavioral battery described below, or maintained in a single cage for a commensurate amount of time without exposure to the testing battery. This was followed immediately by tissue collection.

In order to parse out the importance of metabolism from cholesterol versus metabolism from the prohormone, P, rats received three infusions prior to testing per previous methods (58). The first infusion was either PK11195 or vehicle, followed 20 mins later by a second infusion of either indomethacin or vehicle. Ten mins later rats received a third infusion of either 3α,5α-THP or vehicle and were tested (or not) 10 mins later. This design resulted in eight drug manipulation groups: PK11195/vehicle/vehicle, vehicle/indomethacin/vehicle, PK11195/indomethacin/vehicle, PK11195/vehicle/3α,5α-THP, vehicle/indomethacin/3α,5α-THP, PK11195/indomethacin/3α,5α-THP, vehicle/vehicle/3α,5α-THP, vehicle/vehicle/vehicle. We have previously utilized a similar multiple-infusion strategy with success to assess the necessity of 3α,5α-THP neurosteroidogenesis for affective and social behavior (14,56). However, in the present design, the final infusion consists of vehicle or exogenous 3α,5α-THP in order to further elucidate the mechanisms that underlie effects of central neurosteroidogenesis.

Behavioral Testing

Testing occurred in the same room where screening was conducted daily, so that transport to the core behavioral testing facility would not be a stressor on the day of testing and/or tissue collection. Rats that were tested were put through the battery of tasks described below. All testing apparatus were brightly lit from above with 3 fluorescent bulbs (~200 lux). One of four observers collected all of the behavioral data with the ANY-Maze animal tracking software (Stoelting Co., Wheat Dale, IL) and by hand (as a backup measure). There was greater than 95% concordance between data collected by observers and the ANY-Maze program. As such, the data collected via the ANY-Maze software were used in analyses.

Open Field

Exploration, anxiety, and motor behavior were examined in the open field (81,82). The open field (76 cm × 57 cm × 35 cm) has a 48-square grid floor (6 × 8 squares, 9.5 cm per side) with an overhead light illuminating the central squares (all but the 24 perimeter squares were considered central). As previously reported, rats were placed in the open field and their path was recorded for 5 mins. The numbers of central and total entries were calculated from these data as indices of anti-anxiety and motor behavior, respectively.

Elevated Plus Maze

The elevated plus maze behavior is also used as an index of exploration, anti-anxiety, and motor behavior (82,83). The plus maze was elevated 50 cm off the ground and consisted of four arms (49 cm long and 10 cm wide), with two arms enclosed by walls 30 cm high and two arms exposed. As per previous methods, rats were placed at the juncture of the open and closed arms and the number of entries into, and the amount of time spent on, the open and closed arms was recorded during a 5-min test. Time on the open arms is an index of anti-anxiety and the total number of arm entries is used to assess motor activity.

Social Interaction

The social interaction task assessed exploratory and anxiety behavior associated with interacting with a novel conspecific (82,84). Each member of a pair of rats (1 experimental, 1 stimulus) was placed in opposite corners of an open field (76 cm × 57 cm × 35 cm). The total duration of time that experimental rats engaged an ovariectomized stimulus rat in crawling over and under, sniffing, following with contact, genital investigation, tumbling, boxing, and grooming was recorded during a five-minute test. An ovariectomized rat was utilized as the stimulus animal in order to avoid the possibility of vaginal-cervical stimulation of experimental rats, which might occur if a male had been used as the stimulus animal. Duration of time spent interacting with a conspecific is an index of anti-anxiety behavior.

Paced Mating

Paced mating was utilized over standard mating because of its greater ethological relevance and procedures were carried out as previously reported (56,85). Paced mating tests were conducted in a chamber (37.5 cm × 75 cm × 30 cm), which was equally divided by a partition that had a small (5 cm in diameter) hole in the bottom center, to allow a female free access to both sides of the chamber, but to prevent the larger stimulus male from moving between sides. Females were placed in the side of the chamber opposite the stimulus male. Rats were behaviorally tested for an entire ejaculatory series. The frequency of mounts and intromissions that preceded an ejaculation was recorded. As well, the frequency of lordosis (lordosis quotient=incidence of lordosis/number of mounts), percentage of proceptivity (i.e. hopping, darting, ear wiggling; proceptivity quotient), and percentage of aggression (i.e. vocalizations, defensive postures; aggression quotient) was recorded. Pacing measures included the percentage of times the female left the compartment containing the male after receiving a copulatory stimulus (% exits after mounts, intromissions, and ejaculations) and latencies in seconds to return to the male compartment after these stimuli. The normal pattern of pacing behaviors for percent exits and return latencies to be longer after more intensive stimulation (ejaculations>intromissions>mounts) was observed in the present study.

Tissue Collection

Immediately following testing, trunk blood and whole brains were collected and stored for later measurement of corticosterone, E, P, DHP, and 3α,5α-THP. Trunk blood was centrifuged at 3000 × g for 10 mins and serum was stored in Eppendorf vials at -80 °C. Brains were rapidly frozen on dry ice and stored at -80 °C for approximately three months prior to radioimmunoassay.

Tissue Preparation

Serum was thawed on ice and steroids were extracted as described below. Brains were thawed and infusions sites were examined visually as the midbrain, hippocampus, striatum, and cortex were dissected. Remaining tissue (without cerebellum) was also included as a control “interbrain” region in order to assess the specificity of observed changes in steroid concentration to regions of interest. As we have previously demonstrated site-specific effects of VTA infusions, only those subjects with infusion placement commensurate with a hit to the VTA were included. Sixty-nine rats (24 non-tested and 45 tested) had infusions to sites other than the VTA and were excluded from analyses. This resulted in 17 rats in each condition that were tested and 10 rats in each condition that were non-tested. Circulating and central steroids were extracted as described below.

Radioimmunoassay for Steroid Hormones

Concentrations of corticosterone, P, DHP, 3α,5α-THP, and E were assessed using previously reported methods (56,86). No differences in E concentrations were observed between any groups in any tissue investigated. As such, E levels are not reported. The inter- and intra-assay reliability co-efficients were: corticosterone 0.04 and 0.07, E 0.06 and 0.08, P 0.10 and 0.11, DHP 0.10 and 0.09, and 3α,5α-THP 0.10 and 0.11.

Statistical Analyses

In order to assess conversion of P to its 5α-reduced metabolites, the sum of DHP and 3α,5α-THP concentrations were divided by the P concentration observed in each tissue to yield a DHP+3α,5α-THP:P ratio, per prior methods (87). One-way ANOVAs were used to assess differences in neuroendocrine status between tested and non-tested rats. Two-way ANOVAs, were used to examine effects of 3α,5α-THP inhibitors (vehicle, PK11195 and/or indomethacin) and subsequent 3α,5α-THP infusions (vehicle, 3α,5α-THP) on neuroendocrine and/or behavioral measures within tested and non-tested rat groups. Fisher's Protected Least Significant Difference post-hoc comparisons were used to assess group differences where appropriate. Alpha level for significance was p ≤ 0.05. Trends toward significance are reported when p < 0.10. Power analyses were utilized to verify that all inferential statistics reported were valid with sufficient power.

RESULTS

Endocrine Measures

Among tested rats, infusions of inhibitors decreased, and subsequent 3α,5α-THP infusions enhanced, metabolism of P to its 5α-reduced metabolites, DHP and 3α,5α-THP (Figure 1). Among tested rats, intra-VTA infusion of inhibitors significantly reduced P turnover to its 5α-reduced metabolites in midbrain compared to vehicle [F(3,128)=2.63, p = 0.05], but subsequent 3α,5α-THP infusions to VTA reinstated P turnover in midbrain [F(1,128)=4.55, p < 0.05] and elevated P turnover in hippocampus [F(1,128)=11.29, p < 0.05], diencephalon [F(1,128)=4.11, p < 0.05], and cortex [F(1,128)=11.19, p < 0.05] (Figure 1), but neither interbrain regions nor plasma (Table 1). No significant changes in P turnover were observed in plasma or any brain region examined among tested rats that had cannulae placement that was incongruous with a hit to the VTA (Table 2).

Fig. 1.

Depicts progesterone (P) turnover to its 5α-reduced metabolites, DHP and 3α,5α-THP, in midbrain (top, left), hippocampus (top, right), cortex (bottom, left), and diencephalon (bottom, right) among tested (n=17 per group) rats infused with vehicle, PK11195, and/or indomethacin, followed by subsequent infusions of vehicle (open bars) or 3α,5α-THP (closed bars) to the midbrain VTA. Significant main effect for inhibitor infusions to differ from vehicle infusions (^ p < 0.05). Significant main effect for subsequent 3α,5α-THP infusions to differ from subsequent vehicle infusions (** p < 0.05).

Table 1.

Depicts turnover of progesterone (P) to its 5α-reduced metabolites, dihydroprogesterone (DHP) and 5α-pregnan-3α-ol-20-one (3α,5α-THP) in midbrain, hippocampus, diencephalon, cortex, interbrain, and plasma of non-tested rats (n=10 per group; left) and interbrain and plasma of tested rats (n=17 per group; right) infused with vehicle, PK11195, and/or indomethacin followed by subsequent infusions of vehicle or 3α,5α-THP to the midbrain ventral tegmental area (mean ± SEM). Significant main effect for subsequent 3α,5α-THP infusions to differ from subsequent vehicle infusions.

Progesterone Turnover to 5α-reduced Metabolites (DHP+3α,5α-THP:P ± SEM)
Inhibitor Condition			NON-TESTED RATS						TESTED RATS
Infusate #1	Infusate #2	Infusate #3	Intra-VTA infusions						Intra-VTA Infusions
			Midbrain	Hippocampus	Diencephalon	Cortex	Interbrain	Plasma	Interbrain	Plasma
Vehicle	Vehicle	Vehicle	11.9 ± 1.3	14.8 ± 2.0	11.9 ± 2.8	3.1 ± 0.5	3.0 ± 0.3	2.6 ± 0.7	4.0 ± 0.5	3.6 ± 0.7
PK11195	Vehicle	Vehicle	12.9 ± 2.2	17.2 ± 3.0	8.6 ± 1.0	4.3 ± 0.5	3.9 ± 1.2	2.5 ± 0.9	3.6 ± 0.5	3.9 ± 0.6
Vehicle	Indomethacin	Vehicle	9.2 ± 1.1	11.4 ± 1.7	8.1 ± 1.2	2.8 ± 0.6	2.3 ± 0.5	1.4 ± 0.4	3.6 ± 0.5	3.8 ± 0.6
PK11195	Indomethacin	Vehicle	11.7 ± 1.7	14.8 1.7	6.0 ± 0.8	2.9 ± 0.5	3.2 ± 0.4	2.4 ± 0.7	2.5 ± 0.3	3.6 ± 0.6
Vehicle	Vehicle	3α,5α-THP	16.9 ± 6.4**	31.9 ± 15.5**	9.1 ± 2.5	3.5 ± 0.4**	2.7 ± 0.5	2.7 ± 0.7	3.5 ± 0.5	3.4 ± 0.3
PK11195	Vehicle	3α,5α-THP	16.5 ± 2 1**	29.5 ± 13.1**	12.5 ± 2.3	6.0 ± 0.8**	2.4 ± 0.5	1.8 ± 0.7	2.9 ± 0.3	3.7 ± 0.4
Vehicle	Indomethacin	3α,5α-THP	13.0 ± 0.9**	19.3 ± 3.5**	7.6 ± 1.3	4.3 ± 0.8**	3.8 ± 1.1	2.2 ± 0.8	3.2 ± 0.5	4.1 ± 0.7
PK11195	Indomethacin	3α,5α-THP	22.1 ± 8.9**	34.3± 12.4**	10.0 ± 2.3	4.1 ± 0.7**	3.3 ± 0.4	1.9 ± 0.6	2.7 ± 0.3	4.0 ± 0.5

^**p < 0.05

Table 2.

Depicts turnover of progesterone (P) to its 5α-reduced metabolites, dihydroprogesterone (DHP) and 5α-pregnan-3α-ol-20-one (3α,5α-THP) in midbrain, hippocampus, diencephalon, cortex, interbrain, and plasma of non-tested (n=24) and tested (n=45) rats infused with vehicle, PK11195, and/or indomethacin followed by subsequent infusions of vehicle or 3α,5α-THP to unintentionally-missed sites (mean ± SEM).

Progesterone Turnover to 5α-reduced Metabolites (DHP+3α,5α-THP:P ± SEM)
Inhibitor Condition			NON-TESTED RATS							TESTED RATS
Infusate #1	Infusate #2	Infusate #3	Unintentional Missed Site Controls							Unintentional Missed Site Controls
			n	Midbrain	Hippocampus	Diencephalon	Cortex	Interbrain	Plasma	n	Midbrain	Hippocampus	Diencephalon	Cortex	Interbrain	Plasma
Vehicle	Vehicle	Vehicle	4	8.1 ± 0.5	11.5 ± 2.2	4.3 ± 0.8	4.7 ± 0.8	2.6 ± 0.6	1.9 ± 0.4	5	7.7 ± 3.1	15.5 ± 1.3	6.3 ± 1.1	11.3 ± 2.0	3.0 ± 0.2	4.1 ± 1.5
PK11195	Vehicle	Vehicle	2	10.0 ± 5.4	6.4 ± 2.7	3.4 ± 3.6	4.0 ± 3.5	3.0 ± 0.3	1.7 ± 0.5	7	5.9 ± 0.7	14.6 ± 2.8	9.6 ± 1.1	13.8 ± 1.6	3.5 ± 0.8	5.7 ± 1.1
Vehicle	Indomethacin	Vehicle	0	N/A	N/A	N/A	N/A	N/A	N/A	6	3.7 ± 1.0	13.3 ± 2.2	14.5 ± 5.1	11.3 ± 2.5	2.9 ± 0.3	4.2 ± 0.8
PK11195	Indomethacin	Vehicle	2	9.1 ± 2.3	8.9 ± 4.0	13.6 ± 7.3	4.7 ± 1.8	4.2 ± 0.7	0.9 ± 0.2	7	3.6 ± 0.7	17.2 ± 4.6	12.4 ± 4.3	11.9 ± 0.9	3.4 ± 0.5	3.1 ± 0.8
Vehicle	Vehicle	3α,5α-THP	6	7.6 ± 1.6	16.9 ± 5.4	9.6 ± 2.4	4.7 ± 1.5	2.5 ± 0.4	2.0 ± 0.5	6	5.8 ± 2.7	5.9 ± 0.9	7.9 ± 2.5	10.6 ± 2.1	3.4 ± 0.9	2.9 ± 0.5
PK11195	Vehicle	3α,5α-THP	3	7.8 ± 2.3	16.1 ± 7.3	17.7 ± 7.9	3.2± 1.3	4.6 ± 0.9	3.6 ± 2.0	3	7.7 ± 2.8	16.4 ± 7.5	8.4 ± 0.8	14.5 ± 4.5	2.4 ± 0.5	4.8 ± 1.0
Vehicle	Indomethacin	3α,5α-THP	3	4.0 ± 0.9	8.4 ± 4.4	4.9 ± 1.4	1.8 ± 0.6	4.1 ± 1.3	1.4 ± 0.1	3	6.2 ± 1.3	19.5 ± 10.7	8.0 ± 0.4	16.5 ± 7.7	2.7 ± 0.5	3.1 ± 0.5
PK11195	Indomethacin	3α,5α-THP	4	9.5 ± 0.7	3.4 ± 0.7	20.2 ± 10.4	5.4 ± 1.2	2.2 ± 0.6	2.3 ± 1.4	8	3.5 ± 0.7	19.1 ± 5.3	10.7 ± 2.7	9.9 ± 2.3	2.8 ± 0.4	3.7 ± 0.3

Among non-tested rats, neither infusions of inhibitors to VTA (Table 1), nor those to missed sites (Table 2), significantly reduced P turnover to 5α-reduced metabolites. However, subsequent 3α,5α-THP infusions significantly increased P turnover in the midbrain [F(1,72)=3.75, p = 0.05], hippocampus [F(1,72)=5.42, p < 0.05], and cortex [F(1,72)=7.69, p < 0.05] compared to that of vehicle when infused to the VTA (Table 1) but not when infused to missed sites (Table 2). No 3α,5α-THP enhancement was seen in other brain regions or plasma of non-tested rats (Table 1). Compared to tested rats, turnover of P to its 5α-reduced metabolites was lower in plasma [F(1,214)=25.49, p < 0.05] and cortex [F(1,214)=76.40, p < 0.05], but greater in midbrain [F(1,214)=21.99, p < 0.05], of non-tested rats. Significant differences between tested and non-tested groups were not observed in P turnover in other brain regions (Table 1) nor did groups have different levels of circulatory corticosterone (Table 3).

Table 3.

Depicts plasma corticosterone among non-tested (n=10 per group; left) and tested rats (n=17 per group; right) receiving infusions of vehicle, PK11195, and/or indomethacin followed by subsequent infusions of vehicle or 3α,5α-THP to the midbrain ventral tegmental area (mean ± SEM).

Plasma Corticosterone (μg/dl ± SEM)
Inhibitor Condition			Non-Tested Rats	Tested Rats
Infusate #1	Infusate #2	Infusate #3
Vehicle	Vehicle	Vehicle	1.9 ± 0.3	2.7 ± 0.1
PK11195	Vehicle	Vehicle	2.1 ± 0.2	1.7 ± 0.3
Vehicle	Indomethacin	Vehicle	2.4 ± 0.3	2.2 ± 0.2
PK11195	Indomethacin	Vehicle	2.0 ± 0.3	2.2 ± 0.2
Vehicle	Vehicle	3α,5α-THP	2.2 ± 0.2	2.2 ± 0.2
PK11195	Vehicle	3α,5α-THP	2.3 ± 0.2	2.0 ± 0.3
Vehicle	Indomethacin	3α,5α-THP	2.3 ± 0.2	2.1 ± 0.2
PK11195	Indomethacin	3α,5α-THP	2.2 ± 0.2	2.3 ± 0.2

Behavioral Measures

Overall, measures revealed that intra-VTA infusions of the 3α,5α-THP inhibitors, PK11195 and/or indomethacin, decreased exploratory, anti-anxiety, social, and sexual behaviors when co-administered with vehicle infusions, whereas 3α,5α-THP infusions following inhibitors maintained these behaviors. These effects were not observed among rats receiving infusions to cannulae that were not congruous with a hit to the VTA (Table 4).

Table 4.

Depicts behavior in the open field, elevated plus maze, social interaction and paced mating tasks among rats that received infusions of vehicle, PK11195, and/or indomethacin followed by subsequent infusions of vehicle or 3α,5α-THP to unintentionally-missed sites (mean ± SEM).

Behavior of Unintentional-Missed Site Controls
Inhibitor Condition				Open Field		Elevated Plus Maze		Social Interaction	Paced Mating
Infusate #1	Infusate #2	Infusate #3	n	Number of Central Entries	Number of Total Entries	Open Arm Time (sec)	Number of Total Arm Entries	Interaction Time (sec)	Lordosis Quotient (%)	Proceptivity Quotient (%)	Aggression Quotient (%)	Percent of Exits (%)
Vehicle	Vehicle	Vehicle	5	34 ± 8	264 ± 13	49 ± 19	16 ± 3	85 ± 25	80 ± 20	72 ± 19	0 ± 0	10 ± 10
PK11195	Vehicle	Vehicle	7	42 ± 12	90 ± 33	26 ± 8	11 ± 1	110 ± 29	86 ± 7	46 ± 13	16 ± 9	6 ± 3
Vehicle	Indomethacin	Vehicle	6	42 ± 10	239 ± 47	61 ± 14	16 ± 2	113 ± 17	77 ± 19	24 ± 19	18 ± 18	13 ± 4
PK11195	Indomethacin	Vehicle	7	42 ± 9	231 ± 15	38 ± 14	13 ± 2	100 ± 30	93 ± 5	55 ± 14	19 ± 12	11 ± 6
Vehicle	Vehicle	3α,5α-THP	6	22 ± 9	190 ± 25	12 ± 4	9 ± 2	148 ± 35	81 ± 16	50 ± 13	8 ± 5	13 ± 8
PK11195	Vehicle	3α,5α-THP	3	40 ± 6	279 ± 11	46 ± 42	15 ± 6	60 ± 29	100 ± 0	48 ± 29	4 ± 4	11 ± 6
Vehicle	Indomethacin	3α,5α-THP	3	33 ± 8	161 ± 67	19 ± 19	8 ± 6	51 ± 18	89 ± 11	67 ± 33	0 ± 0	0 ± 0
PK11195	Indomethacin	3α,5α-THP	8	30 ± 6	208 ± 25	61 ± 21	14 ± 2	160 ± 25	83 ± 12	56 ± 14	14 ± 7	18 ± 5

Open Field

Infusions of inhibitors and 3α,5α-THP interacted [F(3,128)=3.46, p < 0.05] due to inhibitors reducing, and 3α,5α-THP infusions increasing, central entries in the inhibitor, but not the vehicle, conditions (Figure 2, top left). There was also a modest effect [F(3,128)=3.42, p < 0.05] for 3α,5α-THP infusions to increase the total number of entries in the open field among inhibitor-infused rats but not those infused with vehicle alone (Subsequent Vehicle: PK11195=216 ± 14, indomethacin=196 ± 17, PK11195 and indomethacin=180 ± 20, vehicle=249 ± 17; Subsequent 3α,5α-THP: PK11195=252 ± 16, indomethacin=229 ± 12, PK11195 and indomethacin=245 ± 18, vehicle=216 ± 11).

Fig. 2.

Rats (n=17 per group) infused with 3α,5α-THP inhibitors (PK11195 and/or indomethacin) to the midbrain VTA demonstrated decreased entries into the central squares of an open field (top, left), time spent on the open arms of an elevated plus maze (top, right), time spent interacting with a conspecific (bottom, left), and frequency of lordosis in paced-mating task (bottom, right). Subsequent 3α,5α-THP infusions reinstated behavior commensurate with vehicle-infused controls in open field (top, left), elevated plus maze (top, right), and paced-mating tasks (bottom, right). Significant main effect for inhibitor infusions to differ from vehicle infusions (^ p < 0.05). Significant main effect for subsequent 3α,5α-THP infusions to differ from subsequent vehicle infusions (** p < 0.05). Significant interaction between vehicle/inhibitor infusions and subsequent vehicle/3α,5α-THP infusions (▼ indicates different from respective non-3α,5α-THP-infused control, p < 0.05).

Elevated Plus Maze

Infusions of inhibitors and 3α,5α-THP interacted [F(3,128)=4.37, p < 0.05], such that inhibitors reduced, and 3α,5α-THP increased, open arm time in the inhibitor but not the vehicle conditions (Figure 2, top right). As well, 3α,5α-THP infusions increased total arm entries among inhibitor-infused rats but not those infused with vehicle alone [F(3,128)=4.493, p < 0.05] (Subsequent Vehicle: PK11195=8 ± 1, indomethacin=12 ± 2, PK11195 and indomethacin=10 ± 1, vehicle=13 ± 1; Subsequent 3α,5α-THP: PK11195=17 ± 1, indomethacin=13 ± 1, PK11195 and indomethacin=16 ± 2, vehicle=13 ± 1).

Social Interaction

Duration of time spent in social interaction with a conspecific was significantly reduced [F(3,128)=3.82, p < 0.05)] among rats infused with PK11195 and/or indomethacin, compared to vehicle-infused rats (Figure 2, bottom left). Infusions of 3α,5α-THP following infusions of inhibitor tended to increase social interaction time in all inhibitor conditions compared to vehicle [F(1,128)=3.15, p < 0.10].

Lordosis Quotients

Infusions of inhibitors significantly reduced [F(3,128)=3.76, p < 0.05], and subsequent 3α,5α-THP infusions significantly enhanced [F(1,128)=13.52, p < 0.05], frequency of lordosis in all inhibitor conditions compared to vehicle control (Figure 2, bottom right).

Proceptivity Quotients

Frequency of proceptive behaviors were significantly reduced by infusions of inhibitors (PK11195=26 ± 7%, indomethacin=20 ± 6%, PK11195 and indomethacin=21 ± 6%, vehicle=58 ± 11%) [F(3,128)=3.12, p < 0.05] and significantly enhanced by subsequent 3α,5α-THP infusions (PK11195=63 ± 10 %, indomethacin=65 ± 9 %, PK11195 and indomethacin=60 ± 10 %, vehicle=68 ± 8 %) [F(1,128)=30.00, p < 0.05] compared to vehicle infusions.

Aggression Quotients

Neither infusions of inhibitors, nor 3α,5α-THP, significantly altered aggression despite an apparent effect for indomethacin to increase aggression compared to controls (PK11195=13 ± 6%, indomethacin=26 ± 7%, PK11195 and indomethacin=15 ± 4%, vehicle=10 ± 4%) and for subsequent 3α,5α-THP infusion to attenuate this effect compared to vehicle infusion (PK11195=12 ± 4 %, indomethacin=7 ± 5 %, PK11195 and indomethacin=17 ± 6 %, vehicle=14 ± 7 %).

Percent Exits

There was no effect of inhibitors either in the absence (PK11195=14 ± 4%, indomethacin=16 ± 5%, PK11195 and indomethacin 11 ± 3%, vehicle=12 ± 4%) or presence of 3α,5α-THP (PK11195=16 ± 3%, indomethacin=16 ± 4%, PK11195 and indomethacin 19 ± 6%, vehicle=15 ± 3%) on the percent of exits from the male compartment to the empty compartment following mating contacts.

DISCUSSION

The present data are the first to provide direct evidence that central formation of P, and subsequent metabolism to 3α,5α-THP, in the VTA are both necessary and sufficient for normative social and affective behavior. Blocking P's formation from cholesterol via PK11195 and/or 3α,5α-THP's formation from DHP via indomethacin, significantly reduced social interaction with a conspecific, lordosis, and anti-anxiety behavior in an open field and elevated plus maze, suggesting that it is formation of 3α,5α-THP that underlies these effects. Reinstatement of 3α,5α-THP in physiological concentration rescued lordosis and anti-anxiety behavior supporting the hypothesis that actions of 3α,5α-THP in the VTA are sufficient for enhancement of these behaviors. As well, our hypothesis that mating-induced enhancement of social and affective behaviors were influenced by enhanced 5α-reduction was supported, given that central inhibitors attenuated P turnover to its 5α-reduced metabolites among mated, but not non-mated, rats and 3α,5α-THP infusions enhanced P turnover to its 5α-reduced metabolites. The relevance of these findings has since been extended to non-sexual social and affective behaviors (14,56).

The present findings have important implications for neuropsychiatric health in people. Notably, the incidence of neuropsychiatric disorders that are characterized by aberrations in social, sexual, and/or affective behavior (such as depression, anxiety, and mood disorders) are greater among women, compared to men (88,89). In women, when progestogen formation is reduced, either via cyclical progestogen decline, during the post-partum, or via menopause, negative mood and affect are more likely to occur (90-93). It is notable that, in people with depression, neuroimaging studies reveal aberrant circulation and neurotransmitter metabolism in midbrain, hippocampus, and frontal cortex compared to healthy controls (94-96). Negative mood states may be influenced by reduced formation of 3α,5α-THP in the brain.

Understanding the mechanisms and behavioral effects of centrally-formed 3α,5α-THP have critical therapeutic implications for social and affective response among people. Formation of 3α,5α-THP may be an important modulator of social behavior in people and may have biphasic effects that are associated with mood-related pathologies. Among women with premenstrual dysphoric disorder (PMDD), irritability coincides with elevations in 3α,5α-THP across the menstrual cycle (26), however, these effects are not observed among non-afflicted women (97,98). This may be partly due to individual differences in sensitivity to 3α,5α-THP's mood-altering effects, given that subunit composition of 3α,5α-THP's GABAergic targets may confer some of its anxiolytic versus anxiogenic effects (99,100). In animal models, protracted social isolation in mice reduces pro-social behavior concomitant with reduced 3α,5α-THP biosynthesis and expression of 5α-reductase mRNA in brain (101-103). Notably, systemic 3α,5α-THP administration can ameliorate the behavioral perturbation in this model (101-103). Formation of 3α,5α-THP may also be important in people. A subset of men and women that were prescribed a 5α-reductase inhibitor as a treatment for alopecia developed depression that was characterized by impaired social relationships and increased anxiety (104). Similar cases of depression have been reported among women prescribed finasteride for hirsutism (105). While, many individuals may utilize therapeutic enzyme-inhibiting drugs, such as finasteride, without untoward effects, some may be particularly susceptible to side effects that potentiate a negative mood state (106). Supporting this notion, polymorphism and mutation at the gene encoding the PK11195 target, TSPO, is observed among some individuals diagnosed with schizophrenia (107). Thus, aberrations in 3α,5α-THP formation may be important factors in the onset of neuropsychiatric disorder.

One mechanism by which therapeutic drugs may mediate neuropsychiatric disorders may also involve formation of 3α,5α-THP. In rodents, effects of systemic or intracerebroventricular administration of antipsychotics or a pharmacological enhancer of TSPO activity, are attenuated by systemic blockade of 3α,5α-THP formation (108). Similarly, a 5α-reductase blocker has been shown to attenuate olanzapine's anti-anxiety effects in rodents (109), supporting a role for 5α-reduction in these processes, despite in vitro data that did not reveal mRNA expression differences in this enzyme in response to some antidepressants (110). Findings of in vitro investigations of clinically-used therapeutics are mixed, with some reports finding that serotonergic anti-depressants can enhance 3α,5α-THP formation without altering activity of rat 5α-reductase, but rather by enhancing activity of 3α-HSD (110), while others report no changes in 3α-HSD activity (111). Indeed, among women with severe premenstrual syndrome, low baseline levels of peripheral 3α,5α-THP predicted enhanced circulating 3α,5α-THP in response to SSRI treatment concomitant with improvement of dysphoric symptoms (113). Conversely, those that had high baseline 3α,5α-THP in circulation responded to SSRI treatment with lower peripheral 3α,5α-THP and did not improve in symptomology (113). As such, the efficacy of some therapeutics may be reliant on their ability to form 3α,5α-THP.

One caveat that must be considered, is the role that cycloxygenase enzyme expression may play in some behavioral effects observed. While, indomethacin is fairly specific for 3α-HSD (80), indomethacin can also inhibit cyclooxygenase enzymes, and non-specific effects on motor behavior have been reported when indomethacin is administered systemically (79). Indeed, prostaglandin products of cyclooxygenase metabolism play an important role in sexual differentiation during development (114) and may be important in mediating sexual function among men (115). In the present investigation, we assessed motor behavior via total entries made in the open field and total arm entries made in the elevated plus maze. Notably, infusions of indomethacin to the VTA did appear to reduce motor activity in the open field by approximately 21%; albeit, this difference was not statistically-significantly and no motor differences were observed on the elevated plus maze. Thus, while we cannot rule out some non-specific effects of indomethacin, motor effects were modest and specific effects on social, affective, and sexual behavior were consistent with those of PK11195.

Together, the findings of the present investigation revealed that P can act as an important modulator of social and affective behavior, partly via formation of its 5α-reduced metabolites in the VTA. Actions of 3α,5α-THP in this region are critical for enhanced expression of these behaviors in naturally, sexually-receptive rats. Further, socially-mediated enhancement of these products in the midbrain VTA may catalyze 5α-reduction in other brain regions (hippocampus, cortex, diencephalon). These findings are clinically-relevant and suggest that therapeutics that can enhance 5α-reduction in midbrain VTA may improve prognosis of sexual, social, and/or affective disorder.

List of Abbreviations

3α-HSD

3α-hydroxysteroid dehydrogenase

3α,5α-THP

5α-pregnan-3α-ol-20-one

TSPO

18 kDa translocator protein

DHP

Dihydroprogesterone

E

Estradiol

P

Progesterone

VTA

Ventral Tegmental Area