Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Pharmacol Biochem Behav. Author manuscript; available in PMC 2010 October 1.
Published in final edited form as:
PMCID: PMC2744290

Infusions of bicuculline to the ventral tegmental area attenuates sexual, exploratory, and anti-anxiety behavior of proestrous rats


Actions of 5α-pregnan-3α-ol-20-one (3α,5α-THP), in the midbrain ventral tegmental area (VTA) modulate sexual receptivity of female rats. Actions of 3α,5α-THP at GABAergic substrates in the VTA are known to modulate consummatory aspects of sexual behavior among rodents, such as lordosis. However, the extent to which GABAA receptors in the VTA are important for appetitive (exploratory, anti-anxiety, social) aspects of sexual-receptivity is not well-understood. Proestrous rats were bilaterally-infused with saline or bicuculline (100 ng), a GABAA receptor antagonist, to the VTA or missed-site control sites. Rats were assessed for exploratory/anti-anxiety (open field/elevated plus maze), social (social interaction), and sexual (paced-mating) behavior. Compared to saline or missed-site controls, intra-VTA bicuculline significantly reduced the number of central entries in an open field, time spent on the open arms of an elevated plus maze, frequency and intensity of lordosis, anti-aggression towards a male, pacing of sexual contacts, and 3α,5α-THP concentrations in midbrain and hippocampus. Bicuculline-infused rats also displayed less affiliation with a novel conspecific, fewer sexual solicitations, and had lower 3α,5α-THP concentrations in diencephalon and cortex, albeit these were not significant differences. Thus, actions at GABAA receptors in the midbrain VTA are essential for appetitive and consummatory aspects of sexual receptivity among rats.

Keywords: Allopregnanolone, Behavioral Estrus, Corticosterone, Hypothalamic-Pituitary-Adrenal Axis, Lordosis, Progesterone, Social Behavior, Stress


Progesterone (P4) plays a critical role in mediating sexual receptivity of female rodents (Powers, 1972) largely through actions in the ventromedial hypothalamus (VMH) and the ventral tegmental area (VTA) of the midbrain (DeBold and Malsbury, 1989; Frye et al. 1992; Malsbury et al., 1977; Pleim et al. 1990; Takahashi and Lisk, 1985). However, within these regions, P4 acts via different mechanisms. There are many intracellular progestin receptors in the VMH via which P4 can alter gene transcription through classical genomic action (Blaustein et al., 1994). Notably, in this region, rodent sexual behavior is also dependent on actions of estradiol (E2; Meisel et al., 1987; Takahashi and Lisk, 1988). In the VTA, very few progestin receptors exist (Warembourg, 1978) and P4 acts through its metabolism to the neurosteroid 5α-pregnan-3α-ol-20-one (3α,5α-THP). Unlike P4, 3α,5α-THP acts at cell membranes of neurotransmitter targets and is dependent on their downstream signal transduction processes to modulate lordosis (the stereotypical female posture in response to male mounting; Frye et al., 2006a). Actions of 3α,5α-THP in the VTA are essential for P4’s lordosis-enhancing effects (Frye et al., 2006a).

One mechanism underlying 3α,5α-THP’s effects in the VTA involves actions at GABAA receptors. GABAergic neurons have been identified using in vivo extracellular and intracellular recordings in rat VTA (Steffensen et al., 1998). GABAergic neurons play a primary role in the local inhibition of mesocorticolimbic dopamine neurons which are plentiful in this region and can modulate GABA release (Kalivas and Duffy, 1995). 3α,5α-THP is the most potent known endogenous modulator of GABAA receptors (Majewska et al., 1986) and can act at these substrates to facilitate consummatory sexual behavior (lordosis; Frye 2001; McCarthy et al., 1995) by enhancing GABAergic function (Twyman and Macdonald, 1992). Further, inhibiting glutamic acid decarboxylase, the enzyme that catalyzes GABA formation from glutamate, in this region reduces lordosis among rats and hamsters (Frye and Vongher, 1999; McCarthy et al., 1995). Thus, actions at GABAA receptors in the VTA are important for consummatory aspects of mating in the female rodent.

In additional to using lordosis as a biosassay, or index of the capacity for steroids to mediate consummatory aspects of sexual behavior, we are also interested in how progestogens mediate appetitive aspects of mating. When in the sexually-receptive phase of the estrous cycle (proestrous), female rats demonstrate less species-typical anxiety-like behavior and conspecific-avoidance, and spend more time in affiliation (Frye and Rhodes, 2008; Mora et al., 1996; Reddy and Kulkarni, 1999). Actions of progestogens at GABAergic substrates in the hippocampus have been implicated such appetitive behaviors. For instance, soporific and anxiolytic effects associated with benzodiazepines (which target GABAA receptors) have long been demonstrated to act in hippocampus (Haefely, 1979; Jahnsen and Laursen, 1981). As well, 3α,5α-THP administration to hippocampus is anxiolytic (Bitran et al., 1991, 2000). While, GABAA receptor activation may underlie affective effects in hippocampus, the role of allosteric action in midbrain for anti-anxiety and associated appetitive sexual behavior is not well-understood. As such, we aimed to assess the necessity of GABAA receptors in the midbrain VTA for appetitive (exploratory, anti-anxiety, social, sexual approach and solicitation) and consummatory (lordosis) aspects of mating behavior. We anticipated that infusion of the GABAA receptor antagonist, bicuculline, to the VTA would attenuate proestrous-typical behavior among female rats.

Materials and Methods

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

Animals and Housing

Adult, intact female Long-Evans rats (N=35) were 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). Rats were group-housed in a temperature- and humidity-controlled room on a 12/12 h reverse light cycle (lights off at 0800 h) with ad libitium access to water and rat chow in their cages.


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; Paxinos and Watson, 1986) under xylazine (12 mg/kg) and ketamine (60 mg/kg) anesthesia. Guide cannulae consisted of modified 23-gauge thin-wall stainless steel needles. Following surgery, rats were monitored for loss of weight, righting response, flank stimulation response, and/or muscle tone (Marshall and Teitelbaum, 1974).

Evaluation of Sexual Receptivity

Estrous cycle phase was determined by daily examination of vaginal epithelium (between 0700–0800 h), per previous methods (Frye et al., 2000; Long and Evans, 1922). Rats with cytology characterized by the presence of many nucleated epithelial cells were considered to be in the proestrous phase of their cycle. During this phase E2 levels are declining, but progestogen levels are high (Feder, 1981; Frye and Bayon, 1999). Rats with proestrous vaginal smears were vaginally masked to prevent alterations in neuroendocrine status that can arise from vaginocervical stimulation (Frye and Bayon, 1999; Meerts and Clark, 2007, 2009; Pfaus et al., 1994) and briefly paired with a sexually-vigorous male. Sexual-receptivity was determined by the response of experimental females to stimulus male investigation. Rats that demonstrated receptive (lordosis) and proceptive behaviors (hopping, darting, ear wiggling) were considered to be in behavioral estrus, while those that exhibited aggressive behaviors (vocalizing, defensive posturing, boxing, avoidance) were considered to not be in behavioral estrus. Only rats that demonstrated both proestrous cytology and sexually-receptive behavior were tested.

Behavioral Testing

Rats in behavioral estrus were tested in the battery of tasks described below. All testing apparatus were brightly-lit from above. All behavioral data were collected with the ANY-Maze data collection program (Stoelting Co., Wheat Dale, IL) and were hand-collected by one of two observers. There was a 97% concordance rate between data that was collected by ANY-Maze and that collected by observers.

Open field

Behavior in the open field has been shown to be an index of exploration, anxiety, and motor behavior (Blizard et al., 1975; Frye et al., 2000). The open field (76 × 57 × 35 cm) had a 48-square grid floor (6 × 8 squares, 9.5 cm/side): there was an overhead light illuminating the central squares (all but the 24 perimeter squares were considered central). Per previous methods, rats were placed in the open field and the path of their exploration was recorded for five-minutes. The number of central, peripheral, and total entries was then calculated from these data as indices of anxiolysis and motor behavior, respectively.

Elevated plus maze

Behavior in the elevated plus-maze has been utilized to assess exploration and anxiety in the past (File, 1993; Frye et al., 2000). The elevated plus-maze was elevated 50 cm off the ground and consisted of four arms (49 cm long and 10 cm wide). Two arms were enclosed by walls 30 cm high and the other two arms were 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 were recorded during a five-minute test. Time spent on the open arms was used as an index of anxiety and the total number of arm entries was a measure of motor activity.

Social interaction

The social interaction task assessed exploratory and anxiety behavior associated with interacting with a novel conspecific (File and Seth, 2003; Frye et al., 2000). Each member of a pair of rats (1 experimental, 1 stimulus) was placed in opposite corners of an open field (76 × 57 × 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 (Frye et al., 2000). An ovariectomized rat was utilized as the stimulus animal in order to avoid the possibility of vaginocervical 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 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 (Erskine, 1985; Frye and Erskine, 1990; Gans and Erskine, 2003; McClintock and Adler, 1978). Paced mating tests were conducted in a chamber (37.5 × 75 × 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 which prevented 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. Behaviors recorded were the frequency of mounts and intromissions that preceded an ejaculation. As well, the frequency (lordosis quotient=incidence of lordosis/number of mounts) and intensity (lordosis rating) of lordosis, quantified by rating of dorsiflexion on a scale of 0–3 (Hardy and DeBold, 1972), was recorded. The percentage of proceptive (i.e. hopping, darting, ear wiggling; proceptivity quotient) and aggressive (i.e. vocalizations, defensive postures; aggression quotient) behaviors prior to contacts was also recorded. Pacing measures included the percentage of times the female left the compartment containing the male after receiving a particular copulatory stimuli (% 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.

Intracranial Drug Administration

Proestrous rats were infused (1 μl, bilaterally) with vehicle (saline; n=12) or the GABAA receptor antagonist, bicuculline (100 ng; n=15). Bicuculline acts by non-competitively, inhibiting ion channel opening of GABAA receptors that are targets for neuroactive steroids (Ueno et al., 1997). Intra-VTA administration of this dose of bicuculline has previously been demonstrated to attenuate lordosis among intact, proestrous or OVX, E2- and P4-primed rats and hamsters (Frye et al., 2006a; Frye and Vongher, 1999).

Tissue Collection

Immediately following testing, trunk blood and whole brains were collected and stored at −80° for later measurement of corticosterone, E2, P4, dihydroprogesterone (DHP), and 3α,5α-THP. Trunk blood was centrifuged at 3000 × g for 10 minutes and serum was stored 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. Assessing brains for endocrine analyses precluded histological analyses. As such, brains were coronally sectioned at the cannulation site so that they could be visually-inspected for placement in the intended region. Eight out of 23 brains in the bicuculline condition presented with cannula placement that was discrepant with a bilateral hit to the VTA as previously reported (Frye and Petralia, 2003; Frye and Seliga, 2003a, b). Brains were then thawed on ice and midbrain, hippocampus, hypothalamus, cortex, were grossly dissected as previously described and the remaining tissue without cerebellum was used as a control (“inter-brain”) (Frye and Rhodes, 2006). Following dissection, steroids were extracted from brain tissue as described below.

Radioimmunoassay for Steroid Hormones

Corticosterone, E2, P4, DHP, and 3α,5α-THP concentrations were measured as described below, using previously reported methods (Choi and Dallman, 1999; Frye and Bayon, 1999; Frye et al., 1996).

Radioactive Probes

[3H]corticosterone (NET 182: specific activity = 48.2 ci/mmol), [3H]E2 (NET-317, 51.3 Ci/mmol), [3H]P4 (NET-208: specific activity=47.5 Ci/mmol), and [3H]3α,5α-THP (used for DHP and 3α,5α-THP, NET-1047: specific activity=65.0 Ci/mmol), were purchased from Perkin Elmer (Boston, MA).

Extraction of Steroids from Serum

Corticosterone was extracted from serum by heating at 60°C for 30 minutes (Choi and Dallman, 1999). E2, P4, DHP, and 3α,5α-THP were extracted from serum with ether following incubation with water and 800 cpms of 3H steroid (Frye and Bayon, 1999). After snap-freezing twice, test tubes containing steroid and ether were evaporated to dryness in a speed drier. Dried down tubes were reconstituted with phosphate assay buffer to the original serum volume.

Extraction of Steroids from Brain Tissues

E2, P4, DHP, and 3α,5α-THP were extracted from brain tissues following homogenization with a glass/glass homogenizer in 50% MeOH, 1% acetic acid. Tissues were centrifuged at 3,000 × g and the supernatant was chromatographed on Sepak-cartridges. Steroids were eluted with increasing concentrations of MeOH (50% MeOH followed by 100% MeOH). Solvents were removed using a speed drier. Samples were reconstituted in 500 μl assay buffer.


The corticosterone antibody (#B3-163, Endocrine Sciences), which typically binds 40–60% of [3H]corticosterone was used in a 1:20,000 dilution. The E2 antibody (E#244, Dr. G.D. Niswender, Colorado State University, Fort Collins, CO), which generally binds between 40% and 60% of [3H]E2, was used in a 1:40,000 dilution. The P4 antibody (P#337 from Dr. G.D. Niswender, Colorado State University) used in a 1:30,000 dilution typically binds between 30% and 50% of [3H]P4. The DHP (X-947) and 3α,5α-THP antibodies (#921412-5, purchased from Dr. Robert Purdy, Veterans Medical Affairs, La Jolla, CA) used in a 1:5000 dilution binds between 40–60% of [3H]3α,5α-THP.

Set-up and Incubation of Radioimmunoassays

The range of the standard curves was 0–4 ng for corticosterone, 0–1000 pg for E2, and 0–8000 pg for P4, DHP, and 3α,5α-THP. Standards were added to assay buffer followed by addition of the appropriate antibody (described above) and [3H] steroid. Total assay volumes were 900 μl for corticosterone, 800 μl for E2 and P4, 950 μl for DHP, and 1250 μl for 3α,5α-THP. All assays were incubated overnight at 4°C, except for corticosterone which incubated at room temperature for 60 min.

Termination of Binding

Separation of bound and free steroid was accomplished by the rapid addition of dextran-coated charcoal. Following incubation with charcoal, samples were centrifuged at 3000 × g and the supernatant was pipetted into a glass scintillation vial with 5 ml scintillation cocktail. Sample tube concentrations were calculated using the logit-log method of Rodbard and Hutt (1974), interpolation of the standards, and correction for recovery with Assay Zap. The inter- and intra-assay reliability co-efficients were: corticosterone 0.05 and 0.11, E2 0.07 and 0.05, P4 0.11 and 0.01, DHP 0.11 and 0.04, and 3α,5α-THP 0.09 and 0.09.


Separate one-way analyses of variance (ANOVAs) were conducted to determine effects of inhibitor infusion (intra-VTA vehicle, intra-VTA bicuculline, or missed site bicuculline) on behavioral and neuroendocrine endpoints. All pairwise contrasts via Fisher’s Protected Least Significant Difference post-hoc tests were conducted to assess group differences. Alpha level for statistical significance was p < 0.05. Trends towards significance were noted in text when p < 0.10.


Behavioral Endpoints

Open Field

Infusions of bicuculline to VTA, but not missed sites, significantly reduced the number of entries rats made into the center of the open field compared to infusions of vehicle [F(2,32) = 5.64, p < 0.05] (Fig. 1, top). Total entries made in the open field did not significantly differ between intra-VTA bicuculline- (272 ± 23) or vehicle-infused rats (315 ± 31) or missed site bicuculline-infused rats (358 ± 25).

Figure 1
Proestrous rats infused with bicuculline (100 ng; n=15) to the ventral tegmental area (VTA) of the midbrain made fewer entries into the center of a brightly lit open field (top) and spent less time on the open arms of an elevated plus maze (bottom) than ...

Elevated Plus Maze

Compared to intra-VTA infusions of vehicle or missed site infusions of bicuculline, intra-VTA infusions of bicuculline significantly reduced the amount of time rats spent on the open arms of the elevated plus [F(2,32) = 3.63, p < 0.05] (Fig. 1, bottom). As well, bicuculline-infused rats made fewer arm entries (9 ± 1) than did vehicle (15 ± 1) or missed site bicuculline-infused control rats (14 ± 2) [F(2,32) = 4.80, p < 0.05].

Social Interaction

Rats infused with bicuculline to the VTA tended to spend less time (76 ± 8 sec) interacting with a novel conspecific compared to those infused with vehicle to the VTA (97 ± 9 sec) and spent significantly less time interacting compared to those infused with bicuculline to missed sites (128 ± 22 sec) [F(2,32) = 4.31, p < 0.05].

Paced Mating

Infusions of bicuculline to the VTA significantly reduced the frequency [F(2,32) = 4.75, p < 0.05] and intensity [F(2,32) = 9.05, p < 0.05] of lordosis among rats compared to infusions of vehicle to the VTA or bicuculline to missed sites (Fig. 2). As well, compared to either vehicle-infused or missed site controls, rats infused with bicuculline to the VTA demonstrated less proceptive behavior (Fig. 3, top), significantly more aggression towards stimulus males [F(2,32) = 3..73, p < 0.05] (Fig. 3, middle), and significantly less pacing of their mating contacts [F(2,32) = 3.42, p < 0.05] (Fig. 3, bottom).

Figure 2
Frequency (top) and intensity (bottom) of lordosis was reduced among proestrous rats receiving intra-ventral tegmental area (VTA) infusions of bicuculline (100 ng; n=15) compared to those infused with vehicle to VTA (n=12) or bicuculline to missed sites ...
Figure 3
Compared to proestrous rats infused with vehicle to the ventral tegmental area (VTA) of the midbrain (n=12) or bicuculline to missed sites (100 ng; n=8), rats infused with bicuculline to VTA (100 ng; n=15) demonstrated fewer proceptive behaviors (top), ...

Neuroendocrine Endpoints

There were no differences between groups in concentrations of E2, P4, or DHP in any tissue examined, nor did plasma corticosterone levels significantly differ (Table 1). Likewise, 3α,5α-THP levels did not significantly differ in plasma, diencephalon, or interbrain. However, there was a significant reduction of 3α,5α-THP among rats infused with bicuculline to the VTA in midbrain [F(2,32) = 3.70, p < 0.05] and hippocampus [F(2,32) = 4.62, p < 0.05] compared to those infused with vehicle to the VTA or bicuculline to missed sites (Table 1). Rats receiving intra-VTA infusions of bicuculline also tended to have lower 3α,5α-THP levels in cortex [F(2,32) = 2.56, p < 0.10], albeit this was not significant (Table 1).

Table 1
Depicts concentrations (mean ± SEM) of estrogen (E2), progesterone (P4), dihydroprogesterone (DHP), and 5α-pregnan-3α-ol-20-one (3α,5α-THP) in serum, midbrain, hippocampus, diencephalon, cortex, and interbrain, ...


The present findings supported the hypothesis that infusion of the GABAA antagonist, bicuculline, to the midbrain VTA would attenuate exploratory, anti-anxiety, and consummatory/appetitive aspects of mating behavior that are typical of proestrous rats. Intra-VTA infusion of bicuculline significantly attenuated anti-anxiety behavior in an open field and an elevated plus maze and reduced duration of interaction time with a novel conspecific compared to control infusions. Bicuculline significantly attenuated consummatory aspects of sexual behavior characterized by lordosis, as well as appetitive sexual behavior processes, such as sexual solicitation, reduction of aggression, and approach-avoidance behavior in the paced-mating paradigm. Notably, these attenuations in behavior never exceeded ~50 % reduction indicating that the importance of alternate mechanisms in these processes. Thus, GABAA receptors in the VTA appear to play an important role in mediation of appetitive and consummatory aspects of mating among rats, albeit, other mechanisms must also be critical for these behaviors.

The present investigation supports and extends prior findings. We have previously observed that inhibition of GABAA receptors in VTA via bicuculline can attenuate enhancement of consummatory aspects of mating, such as lordosis frequency and intensity, among sexually-receptive rodents (Frye and Vongher, 1999; Frye et al., 1993, 2006b). The current investigation confirms these findings and extends them to appetitive measures associated with natural rodent receptivity, including exploratory/anti-anxiety behavior and pacing of sexual contacts. These processes may be involved in many aspects of engagement in motivated behavior given that anxiety must be reduced and social approach must be enhanced in order to locate and engage in copulation with a mate. Others have found that intra-VTA blockade of GABAA receptors via bicuculline can attenuate engagement in some motivated behaviors including opioid-induced feeding (Echo et al., 2002) and alcohol consumption among alcohol preferring rats (Nowak et al., 1998). However, other reports suggest that bicuculline administration to VTA (particularly the anterior region) can be reinforcing (Ikemoto et al., 1997; Xi and Stein, 2000). Together, these data support findings that resolve GABAA receptors to mediate bidirectional reward signals in VTA dependent on endogenous reward state (Lavoilette and van der Kooy, 2001Lavoilette and van der Kooy, 2004). Thus, actions at GABAA receptors in VTA may contribute to many aspects of natural reward and these processes are likely dependent on interactions between endogenous reward systems (such as opioid-dependent processes) and hormonal state.

In the present study, infusions of bicuculline were also associated with some alteration of pregnane neurosteroid formation. In the past, we have found that mating-enhanced 3α,5α-THP formation in brain is not dependent on peripheral steroid production and can be blocked or enhanced by central administration of neurosteroidogenesis inhibitors or enhancers, respectively (Frye et al., 2008, 2009). As such, engaging in paced-mating may trigger de novo synthesis of pregnane neurosteroids in brain. In the current study, the observed reduction of 3α,5α-THP in midbrain and hippocampus among bicuculline-infused rats is intriguing and has several implications. First, it does not seem plausible that central 3α,5α-THP attenuation can be attributable to direct effects of the infusate, bicuculline, particularly given that 3α,5α-THP was not reduced in any tissue among missed site bicuculline-infused controls. Second, reductions in 3α,5α-THP may be indirectly due to actions of bicuculline. We have demonstrated that engaging in paced mating can readily enhance 3α,5α-THP in midbrain and hippocampus (and to a lesser extent in diencephalon and cortex; Frye and Rhodes, 2006; Frye et al., 2007) and bicuculline reduces engagement in mating. As such, reductions in 3α,5α-THP in these regions may be the result of reduced engagement in paced-mating compared to proestrous controls. Third, bicuculline may also have effects on 3α,5α-THP synthesis via actions at a novel sites that underlie neurosteroid formation. For instance, pregnane xenobiotic receptor (PXR) is a promiscuous nuclear transcription factor that is both a target of 3α,5α-THP (Kliewer et al., 2002; Langmade et al., 2006) and is associated with upregulation of CYP3A enzymes which is a rate-limiting step in 3α,5α-THP biosynthesis (Masuyama et al., 2005). Actions of 3α,5α-THP may enhance PXR expression in rodents (Mellon et al., 2008) and this may further enhance neurosteroid biosynthesis. Attenuating 3α,5α-THP actions at GABAA receptors may have effects to dampen PXR enhancement. As well, it could be that bicuculline is a modulator of PXR, given that PXR is a promiscuous xenobiotic receptor. These and other questions regarding this receptor need to be explored. However, bicuculline may also effect neurosteroidogenesis by virtue of being a pharmacological stressor. The midbrain VTA and hippocampus are important regions in the modulation of stress (Herman et al., 2003) and acute stressors can enhance neurosteroid-promoting factors, such as PXR (Narang et al., 2008). Thus stress factors that may underlie bicuculline’s effects must be considered.

An important factor associated with neurosteroid production and pharmacological manipulations of steroid targets is the parasympathestic tone of the organism. Acute stress readily activates the hypothalamic-pituitary-adrenal (HPA) axis and promotes rapid pregnane neurosteroid formation (Drugan et al., 1995; Erskine and Kornberg 1992; Purdy et al., 1991). When elevated 3α,5α-THP can act as a homeostatic modulator in hypothalamus to attenuate corticotrophin releasing hormone formation, thus, dampening HPA arousal (Patchev et al., 1994). Notably, the hippocampus is an important target of stress effects and stress-related disorders are associated with GABAergic dysregulation in this region (Linthorst and Reul, 2008). While, the results of the current investigation do not reveal the mechanism by which intra-VTA actions of bicuculline may alter stress effects mediated by the hippocampus, they do add to what is known regarding the interplay between these structures. Dopaminergic neurons from the VTA project to the hippocampus (Gasbarri et al., 1997; Swanson, 1982) and inhibition of GABAA receptors in the VTA can enhance DA release from projecting neurons (Ikemoto et al., 1997). As well, we and others have found that exposure to lordosis-relevant stimuli is associated with enhancement of dopamine in rodent midbrain, striatum, and/or cortex (Frye, 2001; Meisel et al., 1993). Apart from a direct connection from VTA to hippocampus, a multi-synapse pathway has been proposed wherein GABAergic innervations from the ventral pallidum to the VTA can be activated by the subiculum to create a feedback loop between the hippocampus and the VTA (Lisman & Grace, 2005). We have found that inhibition of 3α,5α-THP in VTA can increase HPA response to stressors associated with task performance in the behavioral battery described (Frye et al., 2008) and others have demonstrated that lesions to the hippocampus can enhance the gluccocorticoid response to open field exposure (Herman et al., 1998; Nyakas et al., 1983). Whether, these differences are due, in part, to GABAergic actions in midbrain and/or hippocampus is an intriguing question. Indeed, electrophysiological and dopaminergic responses to novelty in the hippocampus and midbrain VTA occur concomitantly, indicating that they comprise an important circuit underlying these effects (Lisman & Grace, 2005). Microdialysis studies have also demonstrated that acute stress in rats is associated with increases in extracellular GABA in hippocampus (De Groote and Linthorst, 2007; Bianchi et al., 2003). These data support the notion that GABAergic activity in the VTA may alter stress responses that are mediated by hippocampus.

Notably, there are sex differences in both HPA responding (Luine, 2002) and exploratory behavior of rats (Luine, 2002; Sutcliffe et al., 2007) that are hippocampally-mediated. While, the current investigation found only a modest increase in corticosterone among bicuculline-infused females compared to control females, it is an intriguing question as to whether sex differences would be observed in males. Others have found that bicuculline administration to the VTA can enhance the dopamine-stress response of male rats to restraint (Doherty and Gratton, 2007). As well, stress associated with inter-male aggression results in an enhancement of dopamine production in nucleus accumbens and cortex of threatened rats (Miczek et al., 2008). It is also possible that engaging in the present behavioral paradigm may not have been a salient enough stressor to dissociate between bicuculline- and vehicle-infused females. A recent investigation demonstrates that mild stress associated with exercise in an acute paradigm is not associated with changes in basal corticosterone levels (Droste et al., 2009) and, in our study, the corticosterone levels obtained with bicuclline infusion were scarcely beyond what is typical. Future investigations may aim to utilize males and females with exposure to more salient behavioral stressors.

This investigation demonstrates that GABAergic signaling in the midbrain VTA is important for not only consummatory aspects of rodent mating behavior, but also appetitive aspects of mating that include affective and motivational processes. Given that intra-VTA blockade of GABAA receptors did not abolish any behavior observed, future investigations will aim to assess other factors that may regulate neurosteroid production as well as affective, social, and sexual behaviors.


This research was supported by a grant from the National Institute of Mental Health (MH06769801). We appreciate the help of Danielle Llaneza, Sehee Kim, and Daniel Cusher in collection of behavioral data in addition to assistance from Irene Chin in collection of neuroendocrine data.


Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


  • Bianchi L, Ballini C, Colivicchi MA, Della Corte L, Giovannini MG, Pepeu G. Investigation on acetylcholine, aspartate, glutamate and GABA extracellular levels from ventral hippocampus during repeated exploratory activity in the rat. Neurochem Res. 2003;28:565–73. [PubMed]
  • Bitran D, Foley M, Audette D, Leslie N, Frye CA. Activation of peripheral mitochondrial benzodiazepine receptors in the hippocampus stimulates allopregnanolone synthesis and produces anxiolytic-like effects in the rat. Psychopharmacology. 2000;151:64–71. [PubMed]
  • Bitran D, Hilvers RJ, Kellogg CK. Anxiolytic effects of 3α-hydroxy-5β-pregnan-20-one: endogenous metabolites of progesterone that are active at the GABAA receptor. Brain Res. 1991;561:157–61. [PubMed]
  • Blaustein JD, Tetel MJ, Ricciardi KH, Delville Y, Turcotte JC. Hypothalamic ovarian steroid hormone-sensitive neurons involved in female sexual behavior. Psychoneuroendocrinology. 1994;19:505–16. [PubMed]
  • Blizard DA, Lippman HR, Chen JJ. Sex differences in open-field behavior in the rat: the inductive and activational role of gonadal hormones. Physiol Behav. 1975;14:601–8. [PubMed]
  • Choi S, Dallman MF. Hypothalamic obesity: multiple routes mediated by loss of function in medial cell groups. Endocrinology. 1999;140:4081–8. [PubMed]
  • DeBold JF, Malsbury CW. Facilitation of sexual receptivity by hypothalamic and midbrain implants of progesterone in female hamsters. Physiol Behav. 1989;46:655–60. [PubMed]
  • de Groote L, Linthorst AC. Exposure to novelty and forced swimming evoke stressor-dependent changes in extracellular GABA in the rat hippocampus. Neuroscience. 2007;148:794–805. [PubMed]
  • Doherty M, Gratton A. Differential involvement of ventral tegmental GABAA and GABAB receptors in the regulation of the nucleus accumbens dopamine response to stress. Brain Res. 2007;1150:62–8. [PubMed]
  • Droste SK, Collins A, Lightman SL, Linthorst AC, Reul JM. Distinct, time-dependent effects of voluntary exercise on circadian and ultradian rhythms and stress responses of free corticosterone in the rat hippocampus. Endocrinology. 2009 in press. [PMC free article] [PubMed]
  • Drugan RC, Holmes PV, Scher DM, Luczak S, Oh H, Ferland RJ. Environmentally induced changes in peripheral benzodiazepine receptors are stressor and tissue specific. Pharmacol Biochem Behav. 1995;50:551–62. [PubMed]
  • Echo JA, Lamonte N, Ackerman TF, Bodnar RJ. Alterations in food intake elicited by GABA and opioid agonists and antagonists administered into the ventral tegmental area region of rats. Physiol Behav. 2002;76:107–16. [PubMed]
  • Erskine MS. Effects of paced coital stimulation on estrus duration in intact cycling rats and ovariectomized and ovariectomized-adrenalectomized hormone-primed rats. Behav Neurosci. 1985;99:151–61. [PubMed]
  • Erskine MS, Kornberg E. Stress and ACTH increase circulating concentrations of 3α-androstanediol in female rats. Life Sci. 1992;51:2065–71. [PubMed]
  • Feder HH. Hormones and sexual behavior. Annu Rev Psychol. 1984;35:165–200. [PubMed]
  • File SE. The interplay of learning and anxiety in the elevated plus-maze. Behav Brain Res. 1993;58:199–202. [PubMed]
  • File SE, Seth P. A review of 25 years of the social interaction test. Eur J Pharmacol. 2003;463:35–53. [PubMed]
  • Frye CA. The role of neurosteroids and non-genomic effects of progestins and androgens in mediating sexual receptivity of rodents. Brain Res Brain Res Rev. 2001;37:201–22. [PubMed]
  • Frye CA, Bayon LE. Mating stimuli influence endogenous variations in the neurosteroids 3α,5α-THP and 3α-Diol. J Neuroendocrinol. 1999;11:839–47. [PubMed]
  • Frye CA, Erskine MS. Influence of time of mating and paced copulation on induction of pseudopregnancy in cyclic female rats. J Reprod Fertil. 1990;90:375–85. [PubMed]
  • Frye CA, Mermelstein PG, DeBold JF. Evidence for a non-genomic action of progestins on sexual receptivity in hamster ventral tegmental area but not hypothalamus. Brain Res. 1992;578:87–93. [PubMed]
  • Frye CA, Mermelstein PG, DeBold JF. Bicuculline infused into the hamster ventral tegmentum inhibits, while sodium valproate facilitates, sexual receptivity. Pharmacol Biochem Behav. 1993;46:1–8. [PubMed]
  • Frye CA, Paris JJ, Rhodes ME. Engaging in paced mating, but neither exploratory, anti-anxiety, nor social behavior, increases 5α-reduced progestin concentrations in midbrain, hippocampus, striatum, and cortex. Reproduction. 2007;133:663–74. [PMC free article] [PubMed]
  • Frye CA, Paris JJ, Rhodes ME. Exploratory, anti-anxiety, social, and sexual behaviors of rats in behavioral estrus is attenuated with inhibition of 3α,5α-THP formation in the midbrain ventral tegmental area. Behav Brain Res. 2008;193:269–76. [PMC free article] [PubMed]
  • Frye CA, Paris JJ, Rhodes ME. Increasing 3α,5α-THP following inhibition of neurosteroid biosynthesis in the ventral tegmental area reinstates anti-anxiety, social and sexual behavior of naturally-receptive rats. Reproduction. 2009;137:119–28. [PMC free article] [PubMed]
  • Frye CA, Petralia SM. Lordosis of rats is modified by neurosteroidogenic effects of membrane benzodiazepine receptors in the ventral tegmental area. Neuroendocrinology. 2003;77:71–82. [PubMed]
  • Frye CA, Petralia SM, Rhodes ME. Estrous cycle and sex differences in performance on anxiety tasks coincide with increases in hippocampal progesterone and 3α,5α-THP. Pharmacol Biochem Behav. 2000;67:587–96. [PubMed]
  • Frye CA, Rhodes ME. Progestin concentrations are increased following paced mating in midbrain, hippocampus, diencephalon, and cortex of rats in behavioral estrus, but only in midbrain of diestrous rats. Neuroendocrinology. 2006;83:336–47. [PMC free article] [PubMed]
  • Frye CA, Rhodes ME. The role and mechanisms of steroid hormones in approach/avoidance behavior. In: Elliot AJ, editor. Handbook of approach and avoidance motivation. Mahwah, NJ: Lawrence Erlbaum Associates; 2008. pp. 109–26.
  • Frye CA, Rhodes ME, Petralia SM, Walf AA, Sumida K, Edinger KL. 3α-hydroxy-5α-pregnan-20-one in the midbrain ventral tegmental area mediates social, sexual, and affective behaviors. Neuroscience. 2006a;138 :1007–14. [PMC free article] [PubMed]
  • Frye CA, Seliga AM. Effects of olanzapine infusions to the ventral tegmental area on lordosis and midbrain 3α,5α-THP concentrations in rats. Psychopharmacology. 2003a;170:132–9. [PubMed]
  • Frye CA, Seliga AM. Olanzapine’s effects to reduce fear and anxiety and enhance social interactions coincide with increased progestin concentrations of ovariectomized rats. Psychoneuroendocrinology. 2003b;28:657–73. [PubMed]
  • Frye CA, Van Keuren KR, Rao PN, Erskine MS. Progesterone and 3α-androstanediol conjugated to bovine serum albumin affects estrous behavior when applied to the MBH and POA. Behav Neurosci. 1996;110:603–12. [PubMed]
  • Frye CA, Vongher JM. GABAA, D1, and D5, but not progestin receptor, antagonist and anti-sense oligonucleotide infusions to the ventral tegmental area of cycling rats and hamsters attenuate lordosis. Behav Brain Res. 1999;103:23–34. [PubMed]
  • Frye CA, Vongher JM. Ventral tegmental area infusions of inhibitors of the biosynthesis and metabolism of 3α,5α-THP attenuate lordosis of hormone-primed and behavioural oestrous rats and hamsters. J Neuroendocrinol. 2001;13:1076–86. [PubMed]
  • Frye CA, Walf AA, Petralia SM. In the ventral tegmental area, progestins have actions at D1 receptors for lordosis of hamsters and rats that involve GABAA receptors. Horm Behav. 2006b;50:332–7. [PubMed]
  • Gans S, Erskine MS. Effects of neonatal testosterone treatment on pacing behaviors and development of a conditioned place preference. Horm Behav. 2003;44:354–64. [PubMed]
  • Gasbarri A, Sulli A, Packard MG. The dopaminergic mesencephalic projections to the hippocampal formation in the rat. Prog Neuro-Psychopharma Biol Psychiat. 1997;21:1–22. [PubMed]
  • Haefely W. Involvement of GABA in the actions of neuropsychotropic drugs. Int J Neurol. 1979;13:53–66. [PubMed]
  • Hardy DF, DeBold JF. Effects of coital stimulation upon behavior of the female rat. J Comp Physiol Psychol. 1972;78:400–8. [PubMed]
  • Herman JP, Dolgas CM, Carlson SC. Ventral subiculum co-ordinates situation-specific neuroendocrine and behavioral stress responses. Neuroscience. 1998;86:449–459. [PubMed]
  • Herman JP, Figueiredo H, Mueller NK, Ulrich-Lai Y, Ostrander MM, Choi DC, Cullinan WE. Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary-adrenocortical responsiveness. Front Neuroendocrinol. 2003;24:151–80. [PubMed]
  • Ikemoto S, Kohl RR, McBride WJ. GABAA receptor blockade in the anterior ventral tegmental area increases extracellular levels of dopamine in the nucleus accumbens of rats. J Neurochem. 1997;69:137–143. [PubMed]
  • Ikemoto S, Murphy JM, McBride WJ. Self-infusion of GABAA antagonists directly into the ventral tegmental area and adjacent regions. Behav Neurosci. 1997;111:369–80. [PubMed]
  • Jahnsen H, Laursen AM. The effects of a benzodiazepine on the hyperpolarizing and the depolarizing responses of hippocampal cells to GABA. Brain Res. 1981;207:214–7. [PubMed]
  • Kalivas PW, Duffy P. D1 receptors modulate glutamate transmission in the ventral tegmental area. J Neurosci. 1995;15:5379–88. [PubMed]
  • Kliewer SA, Goodwin B, Willson TM. The nuclear pregnane X receptor: a key regulator of xenobiotic metabolism. Endocr Rev. 2002;23:687–702. [PubMed]
  • Langmade SJ, Gale SE, Frolov A, Mohri I, Suzuki K, Mellon SH, Walkley SU, Covey DF, Schaffer JE, Ory DS. Pregnane X receptor (PXR) activation: a mechanism for neuroprotection in a mouse model of Niemann-Pick C disease. Proc Natl Acad Sci U S A. 2006;103:13807–12. [PubMed]
  • Laviolette SR, van der Kooy D. GABAA receptors in the ventral tegmental area control bidirectional reward signalling between dopaminergic and non-dopaminergic neural motivational systems. Eur J Neurosci. 2001;13:1009–15. [PubMed]
  • Laviolette SR, van der Kooy D. GABAA receptors signal bidirectional reward transmission from the ventral tegmental area to the tegmental pedunculopontine nucleus as a function of opiate state. Eur J Neurosci. 2004;20:2179–87. [PubMed]
  • Linthorst AC, Reul JM. Stress and the brain: solving the puzzle using microdialysis. Pharmacol Biochem Behav. 2008;90:163–73. [PubMed]
  • Lisman JE, Grace AA. The hippocampal-VTA loop: controlling the entry of information into long-term memory. Neuron. 2005;46:703–13. [PubMed]
  • Long JA, Evans HM. Oestrous cycle in the rat and its associated phenomena. Mem Univ Calif. 1922;6:1–146.
  • Luine V. Sex differences in chronic stress effects on memory in rats. Stress. 2002;5:205–16. [PubMed]
  • Majewska MD, Harrison NL, Schwartz RD, Barker JL, Paul SM. Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor. Science. 1986;232:1004–7. [PubMed]
  • Malsbury CW, Kow LM, Pfaff DW. Effects of medial hypothalamic lesions on the lordosis response and other behaviors in remale golden hamsters. Physiol Behav. 1977;19:223–37. [PubMed]
  • Marshall JF, Teitelbaum P. Further analysis of sensory inattention following lateral hypothalamic damage in rats. J Comp Physiol Psychol. 1974;86:375–95. [PubMed]
  • Masuyama H, Suwaki N, Tateishi Y, Nakatsukasa H, Segawa T, Hiramatsu Y. The pregnane X receptor regulates gene expression in a ligand- and promoter-selective fashion. Mol Endocrinol. 2005;19:1170–80. [PubMed]
  • McCarthy MM, Felzenberg E, Robbins A, Pfaff DW, Schwartz-Giblin S. Infusions of diazepam and allopregnanolone into the midbrain central gray facilitate open-field behavior and sexual receptivity in female rats. Horm Behav. 1995;29:279–95. [PubMed]
  • McCarthy MM, Masters DB, Rimvall K, Schwartz-Giblin S, Pfaff DW. Intracerebral administration of antisense oligodeoxynucleotides to GAD65 and GAD67 mRNAs modulate reproductive behavior in the female rat. Brain Res. 1994;636:209–20. [PubMed]
  • McClintock MK, Adler NT. Induction of persistent estrus by airborne chemical communication among female rats. Horm Behav. 1978;11:414–8. [PubMed]
  • Meerts SH, Clark AS. Female rats exhibit a conditioned place preference for nonpaced mating. Horm Behav. 2007;51:89–94. [PubMed]
  • Meerts SH, Clark AS. Artificial vaginocervical stimulation induces a conditioned place preference in female rats. Horm Behav. 2009;55:128–32. [PMC free article] [PubMed]
  • Meisel RL, Camp DM, Robinson TE. A microdialysis study of ventral striatal dopamine during sexual behavior in female Syrian hamsters. Behav Brain Res. 1993;55:151–157. [PubMed]
  • Mellon SH, Gong W, Schonemann MD. Endogenous and synthetic neurosteroids in treatment of Niemann-Pick Type C disease. Brain Res Rev. 2008;57:410–20. [PMC free article] [PubMed]
  • Miczek KA, Yap JJ, Covington HE., 3rd Social stress, therapeutics and drug abuse: preclinical models of escalated and depressed intake. Pharmacol Ther. 2008;120:102–28. [PMC free article] [PubMed]
  • Mora S, Dussaubat N, Díaz-Véliz G. Effects of the estrous cycle and ovarian hormones on behavioral indices of anxiety in female rats. Psychoneuroendocrinology. 1996;21:609–20. [PubMed]
  • Narang VS, Fraga C, Kumar N, Shen J, Throm S, Stewart CF, Waters CM. Dexamethasone increases expression and activity of multidrug resistance transporters at the rat blood-brain barrier. Am J Physiol Cell Physiol. 2008;295:C440–50. [PubMed]
  • Nowak KL, McBride WJ, Lumeng L, Li TK, Murphy JM. Blocking GABAA receptors in the anterior ventral tegmental area attenuates ethanol intake of the alcohol-preferring P rat. Psychopharmacology. 1998;139:108–16. [PubMed]
  • Nyakas C, De Kloet ER, Veldhuis HD, Bohus B. Hippocampal corticosterone receptors and novelty-induced behavioral activity: effect of kainic acid lesion in the hippocampus. Brain Res. 1983;288:219–28. [PubMed]
  • Patchev VK, Shoaib M, Holsboer F, Almeida OF. The neurosteroid tetrahydroprogesterone counteracts corticotropin-releasing hormone-induced anxiety and alters the release and gene expression of corticotropin-releasing hormone in the rat hypothalamus. Neuroscience. 1994;62:265–71. [PubMed]
  • Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. New York: Academic Press; 1986.
  • Pleim ET, Lisciotto CA, DeBold JF. Facilitation of sexual receptivity in hamsters by simultaneous progesterone implants into the VMH and ventral mesencephalon. Horm Behav. 1990;24:139–51. [PubMed]
  • Pfaus JG, Jakob A, Kleopoulos SP, Gibbs RB, Pfaff DW. Sexual stimulation induces Fos immunoreactivity within GnRH neurons of the female rat preoptic area: interaction with steroid hormones. Neuroendocrinology. 1994;60:283–90. [PubMed]
  • Powers JB. Facilitation of lordosis in ovariectomized rats by intracerebral progesterone implants. Brain Res. 1972;48:311–25. [PubMed]
  • Purdy RH, Morrow AL, Moore PH, Jr, Paul SM. Stress-induced elevations of γ-aminobutyric acid type A receptor-active steroids in the rat brain. Proc Natl Acad Sci U S A. 1991;15(88):4553–7. [PubMed]
  • Reddy DS, Kulkarni SK. Sex and estrous cycle-dependent changes in neurosteroid and benzodiazepine effects on food consumption and plus-maze learning behaviors in rats. Pharmacol Biochem Behav. 1999;62:53–60. [PubMed]
  • Rodbard D, Hutt DM. International Atomic Energy Agency. Statistical analysis of radioimmunoassay and immunoradiometric assays: a generalized, weighted iterative, least squares method for logistic curve fitting. Symposium on Radioimmunoassay and Related Procedures in Medicine; New York: Uniput; 1974. pp. 209–33.
  • Steffensen SC, Svingos AL, Pickel VM, Henriksen SJ. Electrophysiological characterization of GABAergic neurons in the ventral tegmental area. J Neurosci. 1998;18:8003–15. [PubMed]
  • Sutcliffe JS, Marshall KM, Neill JC. Influence of gender on working and spatial memory in the novel object recognition task in the rat. Behav Brain Res. 2007;177:117–25. [PubMed]
  • Swanson LW. The projections of the ventral tegmental area and adjacent regions: a combined fluorescent retrograde tracer and immunofluorescence study in the rat. Brain Res Bull. 1982;9:321–53. [PubMed]
  • Takahashi LK, Lisk RD. Diencephalic sites of progesterone action for inhibiting aggression and facilitating sexual receptivity in estrogen-primed golden hamsters. Endocrinology. 1985;116:2393–9. [PubMed]
  • Takahashi LK, Lisk RD. Dual progesterone action in diencephalon facilitates the induction of sexual receptivity in estrogen-primed golden hamsters. Physiol Behav. 1988;44:741–7. [PubMed]
  • Twyman RE, Macdonald RL. Neurosteroid regulation of GABAA receptor single-channel kinetic properties of mouse spinal cord neurons in culture. J Physiol. 1992;456:215–45. [PubMed]
  • Ueno S, Bracamontes J, Zorumski C, Weiss DS, Steinbach JH. Bicuculline and gabazine are allosteric inhibitors of channel opening of the GABAA receptor. J Neurosci. 1997;17:625–34. [PubMed]
  • Warembourg M. Radioautographic study of the rat brain, uterus and vagina after [3H]R-5020 injection. Mol Cell Endocrinol. 1978;12:67–79. [PubMed]
  • Xi ZX, Stein EA. Increased mesolimbic GABA concentration blocks heroin self-administration in the rat. J Pharmacol Exp Ther. 2000;294:613–9. [PubMed]