The midbrain VTA is known for its actions for motivated responses, and mating can be considered such a motivated behavior. Mating responses of females are quantified with measures of lordosis, proceptivity, and aggression. Lordosis, the necessary posture of female rodents for mating, can be quantified in the laboratory as the number of such responses by the female as a ratio (or quotient; lordosis quotients) of the attempts by the male. Other behaviors, such as proceptivity (courtship behaviors; proceptivity quotients) or aggression (rejection of males’ advances; aggression quotients) can be concurrently assessed with lordosis. As well, in our laboratory, we typically assess other behaviors beyond those directly related to mating, but those that may have consequences for successful reproduction, such as exploration, reductions in fear/anxiety, and social behavior with conspecifics (for review see Frye, 2009). Mating is a motivated behavior that is only observed under appropriate endocrine and environmental contexts, and one in which the brain circuitry necessary for it to occur (namely in the hypothalamus and midbrain for female rodents), and may modify its expression (e.g., corticolimbic structures), are becoming better characterized (DeBold and Malsbury, 1989; Frye and Walf, 2008; Pfaff et al., 2008; Frye, 2011).

By utilizing this behavioral response of mating as a bioassay, we have been able to determine that allopregnanolone, from both metabolism of circulating progesterone, and biosynthesis in the midbrain, in the midbrain VTA is necessary and sufficient for mating (reviewed recently in Frye, 2011). Requisite enzymes and proteins for metabolism and biosynthesis of allopregnanolone are expressed in the midbrain VTA as well as in corticolimbic regions that may be involved (Cheng and Karavolas, 1975; Li et al., 1997; Furukawa et al., 2002; Frye, 2011; Frye et al., 2013a). Observations of age-related changes in reproductive behaviors and timing of reproductive senescence among female rats suggest that reductions in capacity to form allopregnanolone in the midbrain may be involved (Walf et al., 2011). Genetic knockout of 5α-reductase in female mice lowers allopregnanolone levels in the midbrain and attenuates lordosis during proestrous (when females typically have their highest levels of allopregnanolone, coincident with mating), and following ovariectomy and progesterone administration (Koonce and Frye, 2014). 5α-reductase knockout mice have normative responses to allopregnanolone administration. In addition to these findings that suggest the importance of progesterone metabolism in the midbrain for mating, there are data in support of the role of allopregnanolone synthesis in the VTA for mating. Antagonists of TSPO, P450scc, and 3β-HSD, delivered directly to the midbrain VTA, of receptive rats attenuates lordosis similarly as inhibitors of metabolism by 5α-reductase and 3α-HSD (reviewed in Frye, 2011; Frye et al., 2013a). As well, agonists of TSPO can have similar actions as allopregnanolone to OVX and ADX rats to increase midbrain levels of allopregnanolone and lordosis (reviewed in Frye, 2011; Frye et al., 2013a). Together, these approaches have suggested the importance of allopregnanolone, from both metabolism and biosynthesis, in the midbrain VTA for mating.

In addition to being a measure of interest, mating can induce allopregnanolone formation in the nervous system, and, thereby, can be considered a manipulation as well. Among proestrous female rats that engage in mating with a male, there is a rapid increase in allopregnanolone levels in the midbrain; this same pattern of allopregnanolone synthesis is not observed with the smell, or site, of a sexually-experience male, or a female conspecific (Frye and Bayon, 1999; Frye et al., 2007). Notably, allopregnanolone levels are higher following “paced mating” compared to a standard mating task (Frye, 2001a,b, 2009, 2011; Frye et al., 2007, 2014b). Paced mating is considered a semi-naturalistic mating paradigm as compared to a standard mating paradigm, which is typically performed in a small chamber in a laboratory (e.g., a 10 gallon aquarium). Paced mating is considered closer to the natural experience because the chamber is larger and divided with an entry only a female can transverse to get to the other side of the chamber; as such, female rats can control the timing of (i.e., “pace”) their mating contacts with males, which is a critical part of the natural response in the wild and to enhance fertility and fecundity (Frye and Erskine, 1990). Even in the situation that females are tested in a large, paced mating chamber in the laboratory, but do not spontaneously pace, or show a low pacing response (but the same number of mounts by the male), there are lower levels of allopregnanolone in the midbrain compared to females that do show the pacing response (Frye and Rhodes, 2006). These data support the notion that mating can induce allopregnanolone synthesis; albeit, a question is the role of other reproductively-relevant behaviors, which may precede or follow mating, for allopregnanolone synthesis.

Reproductively-relevant behaviors are those that may improve reproductive success. For example, some of these reproductively-relevant behaviors are those that include increased exploration and reduced anxiety that would promote females leaving the natal nest and traversing a complex and novel environment to encounter other female conspecifics and potential mates for the first time. To address this in our laboratory, paced mating as well as measures of exploration (e.g., open field), anxiety (e.g., elevated plus maze), or social interaction with another female are assessed in a short battery of these tasks. Paced mating itself, or immediately following this battery of tasks, increases allopregnanolone synthesis in the midbrain, compared to testing in the battery without mating (Frye et al., 2007, 2014b). Together, these data support that mating can be utilized as a measure of allopregnanolone’s actions as well as a way to manipulate allopregnanolone levels in the midbrain. This model has then been used to assess the mechanisms of allopregnanolone, with a focus on non-traditional actions for mating and reproduction-relevant behaviors.

The VTA has rich innervation of dopamine targets and some of allopregnanolone’s actions in the VTA for mating may involve these targets, as well as GABA and glutamate. Progestogens can increase release of GABA, dopamine and glutamate (Lévesque and Di Paolo, 1990; Frye et al., 2000; Frye, 2001a,b). High levels of progestogens enhance number, density, and affinity of GABA_A receptors, coincident with enhancing lordosis (Mascó et al., 1986; Wilson, 1992; Frye and Vongher, 1999). There are D₁ receptors on dopaminergic cell bodies and GABAergic terminals as well as NMDARs (Stoof and Kebabian, 1984; Bayer and Pickel, 1991; Willick and Kokkinidis, 1995). Greater GABA input onto GABA_A receptors that are located on GABAergic interneurons in the VTA mitigate inhibitory actions of these cells on dopamine cell bodies, thereby increasing dopamine release (from cell body and dendrites; Churchill et al., 1992). Excitation of D₁ receptors on GABAergic afferents in the VTA increases GABA release (Kalivas and Duffy, 1995). Antagonists of D₁ or GABA_A reduce allopregnanolone-facilitated lordosis when administered to the VTA and the opposite pattern is observed with agonists of D₁ or GABA_A (Frye et al., 2004; Sumida et al., 2005; Frye and Paris, 2009). Furthermore, antagonists of GABA_A receptors to the VTA reduce allopregnanolone-facilitated lordosis, and the potentiation of this response by a D₁ agonist co-administered to the VTA (Frye et al., 2006c). A role of N-methyl-D-aspartate receptor (NMDARs) is also suggested in this pathway. In support, D₁ expressing GABAergic terminals, synapse on dopaminergic cell bodies that express both GABA_A receptors and NMDARs (Bayer and Pickel, 1991; Willick and Kokkinidis, 1995). Infusions of a NMDAR antagonist to the VTA increases allopregnanolone-facilitated reproductive responding of female rodents (Petralia et al., 2007; Frye and Paris, 2011). Together these findings suggest that allopregnanolone’s actions for reproductive responding in the VTA may be related to reductions in tonic inhibition of dopamine neurons in this region, involving actions of GABA_A, D₁, and NMDARs here. Additional studies have suggested downstream pathways for these receptors, including, including activity of G-proteins, adenylyl cyclase, phospholipase C and protein (a discussion of which is beyond the scope of this review, but can be found in Frye and Walf, 2008). Moreover, the functional role of membrane targets of the progestogens, such as the membrane progestin receptors, for reproductive indices have been shown across aquatic species and terrestrial mammals (Tokumoto, 2012; Tokumoto et al., 2012; Frye et al., 2013b, 2014c; Pang et al., 2013; Petersen et al., 2013; Schumacher et al., 2014); the extent to which there are interactions between these ionotropic and metabotropic targets in the VTA is of continued interest. A microarray analysis of gene expression changes in the midbrain of proestrous rats that had been paced mated or not confirmed the role of the targets involved in allopregnanolone metabolism and biosynthesis, as well as these neurotransmitter targets, but also revealed a novel target of interest, the pregnane xenobiotic receptor (PXR; Frye and Walf, 2008; Frye, 2009). The findings to date about this novel target in the midbrain are described as follows.

A recent focus has been on characterizing the role of a promiscuous nuclear 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. PXR has well-known metabolic and clearance actions in the traditional organs for metabolism and excretion, such as the liver, kidneys, intestines, and the blood-brain barrier (Geick et al., 2001; Dussault and Forman, 2002; Kliewer et al., 2002; Francis et al., 2003; Bauer et al., 2004, 2006; Xu et al., 2005; Harmsen et al., 2007; Ma et al., 2008; Zhang et al., 2008; Ott et al., 2009). It is considered a promiscuous nuclear receptor with a long list of molecules that it positively modulates (including several steroids, and allopregnanolone) and much fewer molecules that are negatively modulated. Although early work on understanding the role of PXR outside of the liver and other excretory organs in the body was focused on the blood-brain-barrier, several laboratories, including our own, have demonstrated its expression in the brain proper (e.g., in rodents, rabbits, pigs, and humans; Bauer et al., 2004; Lamba et al., 2004; Marini et al., 2007; Mellon et al., 2008; Frye, 2011). In considering its role for metabolism and xenobiotic (including steroid) clearance in the liver, we sought to determine PXR’s functional effects related to allopregnanolone production and/or action. PXR protein and mRNA was expressed in the midbrain of proestrous rats, with higher expression (determined by western blots) in female rats in proestrous versus those in diestrous or male rats (Frye et al., 2012, 2013b), suggesting a possible role of ovarian steroids (estradiol, progesterone) and/or pregnane neurosteroids. These studies were correlational in nature and demonstrated a relationship between hormonal milieu and sex differences for expression of PXR.

Next, studies investigated manipulations of PXR in the midbrain for functional effects, including lordosis. Positive modulators of PXR, such as allopregnanolone, other pregnane steroids (3β,5α-THP, 3α,5β-THP), and RU486, when infused to the VTA, enhanced lordosis of OVX, estradiol-primed rats (Frye, 2011). However, these findings are tempered by the known promiscuity of PXR. Follow-up studies utilized a pharmacogenetic tool (antisense oligodeoxynucleotides, AS-ODNS) to reduce expression of PXR in the midbrain VTA to further understand functional outcomes (Frye, 2011; Frye et al., 2012, 2013a, 2014a,b). Investigations of the role of PXR, by using this PXR knockdown approach, for mating-induced neurosteroidogenesis and functional effects are ongoing, and some key findings are described as follows.

The role of the PXR for biosynthesis of allopregnanolone in the brain has been investigated. An approach that was utilized to investigate this was to compare the capacity of rats with peripheral glands removed (e.g., the ovaries and/or the adrenal glands) to produce allopregnanolone in the midbrain following mating. In comparing rats that were OVX or OVX/ADX, estradiol-primed and behaviorally tested in the paced mating task to non-tested controls, there was a robust increase in midbrain levels of allopregnanolone, particularly among the OVX rats, with paced mating (Figure 1). This effect was attenuated when rats were administered PXR AS-ODNs to the midbrain VTA (Figure 1). Moreover, comparisons of rats that are paced mated and in different hormonal states (proestrous, OVX, OVX/ADX) and administered saline vehicle or PXR AS-ODN infusions to the midbrain VTA corroborate these findings. Administration of PXR AS-ODNs to the midbrain reduces allopregnanolone levels in the midbrain following mating across these hormone conditions (Figure 2). These data suggest a role of PXR for mating-induced allopregnanolone secretion in the midbrain VTA.

A question is how PXR may interact with other downstream factors recognized to be involved in neurosteroidogenesis. Investigation of this question has begun by assessing the role of TSPO, given that this is one rate-limiting factor for allopregnanolone synthesis in the brain. Inhibiting TSPO with PK11195 reduced allopregnanolone in the midbrain and lordosis, an effect that could be reversed with allopregnanolone replacement, but not when AS-ODNs and allopregnanolone were co-administered. AS-ODNs blocked actions of FGIN 1-27 for lordosis and allopregnanolone levels among proestrous > OVX > OVX/ADX rats. Together, these data support the notion that PXR may be upstream of TSPO. Investigations of the regulation of other related factors are underway.

Although these data suggest that PXR is important for the synthesis of allopregnanolone in the midbrain, a related research question is the downstream factors for allopregnanolone’s actions. PXR AS-ODNs to the VTA, but not nearby midbrain sites, blocks reproductive responding among receptive rats associated with estrous cycle increases or following estradiol- and progestogen-administration to OVX rats (Frye et al., 2012, 2013a, 2014a). Moreover, knocking down expression of PXR in the midbrain VTA attenuates actions of allopregnanolone via NMDA and/or GABA_A receptors for lordosis (Figure 3). That there were some differences noted across hormonal milieu in this study, suggestive of a role of allopregnanolone biosynthesis, follow-up questions would include capacity for allopregnanolone biosynthesis in the brain as well as responses to allopregnanolone administration across these groups.

Another area of interest is the role of PXR for other socially-relevant behaviors. We have traditionally utilized studies such as those described above to ascertain mechanisms of allopregnanolone using lordosis in a mating task as one endpoint. Mating is typically assessed after other measures of behaviors that may support reproduction (i.e., reproductively-relevant behaviors, such as exploration, anxiety, and pro-social behavior) that allopregnanolone mediates (Frye, 2011; Frye et al., 2012, 2013a,b), perhaps through its actions at GABA_A receptors and NMDARs (Frye and Paris, 2009, 2011). In comparing the extent to which PXR AS-ODNs reduce such behaviors, we have consistently noted that the most robust effects are for lordosis quotients, followed by other socially-relevant measures (aggression/rejection during the mating task, and social investigation of a female conspecific), and then affective measures (open arm exploration in the plus maze) and then exploratory/ambulatory behavior (open field entries made; Figure 4). These data suggest overall that manipulations of PXR are most salient for socially-relevant behaviors, and that the midbrain infusions of such drug manipulations are not associated with non-specific effects of ambulatory behavior.

These comparisons suggest the specificity of the response as well as brain targets outside of the midbrain VTA. Among receptive rats, mating-induced allopregnanolone synthesis is observed in the midbrain as well as corticolimbic structures (hippocampus, prefrontal cortex) and the hypothalamus (Frye et al., 2006a, 2007). PXR AS-ODNs to the midbrain VTA of receptive rats have the most salient effects to reduce allopregnanolone in the midbrain, but reductions are also observed in the hippocampus (Frye et al., 2013a, 2014a). Interestingly, PXR AS-ODNs to the midbrain VTA also reduce levels of the growth factor, brain-derived neurotrophic factor (BDNF), in the hippocampus coincident with differences in behavior (Frye et al., 2014b). Allopregnanolone has actions on BDNF as well as cognitive performance of rodents (Nin et al., 2011; Frye et al., 2013a; Bali and Jaggi, 2014). As such, the extent to which PXR is a target of allopregnanolone beyond the midbrain to corticolimbic structures is of great interest. Indeed, genetic knockout of a related nuclear receptor known for its actions in the liver, the liver X receptor, increases anxiety-like behavior of mice and alters GABAergic function in the hypothalamus, as well as may play a protective role in a Parkinson’s disease mouse model (Dai et al., 2012; Tan et al., 2012). Thus, we consider that allopregnanolone may have a role via PXR in the midbrain and beyond for neural and behavioral plasticity.

We have begun characterizing the role of lifelong knock down of PXR as well as species similarities/differences, using PXR knockout (KO) rats and mice. Progesterone administration produced similar rates of lordosis as observed among proestrous wildtype (WT), but not PXRKO, rats; both WT and PXRKO rats responded to allopregnanolone administration with increased lordosis (Figure 5). The same pattern was observed among WT and PXRKO mice, suggesting species similarities in this mechanism (Figure 6). These data corroborate what has been observed with PXR knockdown in the VTA of rats to reduce lordosis of receptive rats, but show that PXRKO rats can respond to allopregnanolone administration (unlike what has been observed with allopregnanolone infusions to the VTA following PXR AS-ODN infusions; Frye et al., 2014a). These results are promising in that they suggest a specific deficit in synthesis of allopregnanolone, rather than binding of allopregnanolone as just one of many, many positive modulators of this promiscuous nuclear receptor. However, it is not known what the capacity for mating-induced allopregnanolone, and whether there are similar brain targets, is in these rodent models at this time. As well, a typical concern with models of whole body and brain knockout of a gene throughout development is the potential for compensatory mechanisms. Studies are ongoing to characterize these animal model resources further.

Of interest is whether there are other behavioral phenotypes in the PXRKO rats and mice to consider. There are no apparent differences in their homecage behavior, and systematic analyses are underway to assess other behavioral endpoints. A prediction, based upon the data with PXR AS-ODNs, is that the most salient effects of PXR knockout may be for reproductive measures (as supported by data in Figures 5 and 6) or social behaviors. Indeed, a pilot assessment of WT and PXRKO mice in the mirror maze supports this notion. The mirror maze is a behavioral assessment of acute changes in rodents’ responses to observations of their own image in a mirror (Houri, 1986; Lamberty, 1998). This task, like several others considered to be an index of affective responsing (e.g., elevated plus maze), is considered a free-choice conflict task in which the time spent by the rodent in the mirrored section of a cubed chamber is compared to the time spent away from the mirrors in an adjoined alleyway without mirrors (Henderson et al., 2004; Frye et al., 2006b). We have utilized this task to assess the role of allopregnanolone and other steroid targets, including the androstane equivalent of allopregnanolone (3α-androstanediol; Frye et al., 2006b, 2008; Walf et al., 2009). In initial assessments of female WT and PXRKO mice during the proestrus phase of the estrous cycle, we noted an approximately 15% increase in time spent in the mirror chamber among the PXRKO mice (197 s) than in the WT mice (172 s). This pattern is opposite to what has been noted with PXR knockdown or knockout for interaction with a mate or conspecific (as described above). As well, female mice with knockout of estrogen receptor beta, which may be another important factor in allopregnanolone synthesis, but not progestin receptor, respond poorly in this task. These data in the mirror maze are interesting as they suggest a role of PXR for mediating responses to another socially-relevant stimuli, the rodents’ own image in a mirror, beyond a mate (as in the paced mating task) or another female conspecific (as in the social interaction task). Although a focus has been on actions of allopregnanolone in the midbrain VTA, how the understanding of these novel targets in this region can be extended elsewhere in the CNS relevant for clinical conditions is of continued interest.