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BOR - Papers in Press, published online ahead of print September 1, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.033126
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BIOLOGY OF REPRODUCTION 72, 33–41 (2005)
DOI: 10.1095/biolreprod.104.033126
© 2005 by the Society for the Study of Reproduction, Inc.

GABAergic Integration of Progesterone and Androgen Feedback to Gonadotropin-Releasing Hormone Neurons1

Shannon D. Sullivan, and Suzanne M. Moenter2

Departments of Internal Medicine and Cell Biology, University of Virginia, Charlottesville, Virginia 22908


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Steroid feedback regulates GnRH secretion and previous work has implicated gamma-aminobutyric acid (GABA)ergic neurons as a mediator of these effects. We examined GABAergic postsynaptic currents (PSCs) in green fluorescent protein-identified GnRH neurons from mice exposed to different steroid milieus in vivo. Adult mice were ovariectomized and treated with estradiol (OVX+E, controls) or E plus progesterone (P, OVX+E+P). P decreased PSC frequency, a presynaptic effect, and PSC size, which could be via pre- and/or postsynaptic mechanisms. In contrast, dihydrotestosterone (DHT, OVX+E+DHT) increased both GABAergic PSC frequency and size in GnRH neurons. Tetrodotoxin (TTX), which eliminates action-potential-dependent presynaptic effects, did not alter frequency, suggesting DHT may have increased PSC frequency by increasing connectivity between GABAergic and GnRH neurons. TTX reduced PSC size below control values, indicating DHT may augment presynaptic GABA release but inhibits the postsynaptic GnRH neuron response. In mice treated with both P and DHT (OVX+E+P+DHT), PSC frequency and size were similar to controls, suggesting these steroids counteract one another. These results demonstrate GABAergic neurons participate in integrating and conveying steroid feedback to GnRH neurons, defining a potential central mechanism for steroid regulation of GnRH neurons during the reproductive cycle, and providing one possible mechanism for increased activity of these cells in hyperandrogenic females.

DHT, feedback, GABA, GnRH, neurotransmitters, progesterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GnRH neurons are the central regulators of fertility and are controlled by steroid feedback. GnRH release must occur in discrete pulses separated by periods of little to no secretion to maintain pituitary function [1], and pulse patterns must vary across the reproductive cycle to differentially regulate release of the two gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) [2, 3]. Low GnRH pulse frequency favors FSH release, whereas high pulse frequency stimulates LH [3]. Progesterone, produced during the luteal phase of the cycle, provides negative feedback to lower GnRH/LH pulse frequency [4, 5]; low-frequency GnRH pulses continue into the early follicular phase, allowing an FSH rise that is crucial for ovarian follicular recruitment and maturation. Androgens also provide feedback to GnRH neurons: in adult male rodents, androgens impose negative feedback [6]. In contrast, data from adult women [7, 8] and from models of prenatal androgenization in primates, sheep, and rodents [9–11] suggest that, in females, excess androgen may disrupt communication of negative feedback signals from progesterone and/or independently stimulate GnRH release.

The central mechanisms for steroid feedback are poorly understood. Steroids may act directly on GnRH neurons, via activation of nuclear receptors expressed in these cells. Convincing evidence in vivo, however, exists only for expression of the beta isoform of the estradiol receptor in rats [12, 13]; in mice, this message has been reported [14] but the protein not yet been detected [15]. Likewise, neither androgen [16] nor progesterone receptors [17] have been detected in GnRH neurons except in a small subpopulation of cells in the guinea pig [18].

Alternatively, steroid feedback may be conveyed to GnRH neurons transsynaptically via steroid-sensitive neurons. A substantial body of work supports gamma-aminobutyric acid (GABA) action on GABAA, a subtype of GABA receptor, receptors (GABAAR) as a possible mediator [19]. GABAergic afferents provide a major anatomical and functional input to GnRH neurons [20–24], and hypothalamic GABAergic neurons are known to express progesterone [25] and estrogen [26] receptors. The role of GABAAR activation in the control of GnRH neurons is controversial. Inhibition is suggested by observations of an inverse correlation between GABA and LH levels in rats and sheep [27–29], and increased LH release or advanced puberty following hypothalamic application of GABAA antagonists or blockade of GABA synthetic enzymes in rats and primates [30–33]. In rat explants treated in vitro, both inhibition and stimulation of GnRH release in response to GABAAR antagonism have been noted, depending on age [34] or explant boundary [35]. In the above studies, it is important to bear in mind that the afferent neuronal circuitry controlling GnRH release is also subjected to the pharmacological treatment; thus, the possibility that the inhibition or stimulation observed may be indirect cannot be eliminated. Of interest in this regard, inducible localized increases in GABA near GnRH nerve terminals in the median eminence in vivo stimulated the hypothalamic-pituitary-ovarian axis in rats [36].

GABAAR activation opens an intrinsic chloride ion channel; the direction of chloride flow depends on resting membrane potential and intracellular chloride levels. Unlike most mature neurons, GnRH neurons maintain high intracellular chloride [37]. Direct studies of GnRH neurons have shown both depolarizing and hyperpolarizing actions of applied GABA [37, 38] and inhibitory and stimulatory roles for endogenous GABA [39, 40]. Studies of alterations in GABAergic drive to GnRH neurons induced by negative energy balance and neuromodulators involved in conveying metabolic signals to the reproductive endocrine system indicate a direct physiological correlation between increased GABAAR-mediated drive to these cells and increased reproductive activity [11, 23, 24].

The above debate notwithstanding, the advent of transgenic models in which GnRH neurons can be visualized in living brain slices [39, 41, 42] allows direct testing of GABAergic mechanisms by which steroid feedback may be communicated to GnRH neurons. The whole-cell patch-clamp technique was used to record from GnRH neurons in brain slices from ovariectomized (OVX) adult female mice treated with physiological levels of steroid hormones in vivo. Spontaneous GABAergic postsynaptic currents (PSCs) were recorded to examine overall changes in GABAAR-mediated signaling to GnRH neurons; miniature GABAergic postsynaptic currents (mPSCs), which are action-potential-independent, were recorded to begin to examine whether changes in spontaneous PSC properties were due to pre- and/or postsynaptic mechanisms. Frequency and size of these GABAAR-mediated currents were compared to explore how progesterone and/or androgens may alter GABAergic signaling to GnRH neurons.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Adult transgenic female mice in which green fluorescent protein is genetically targeted to GnRH neurons [42] were used. Animals were housed in groups of 3–5 on a 14L:10D light cycle with lights on at 0500 h Eastern Standard Time and maintained on standard rodent chow (7012; Harlan, Bartonsville, IL) and water ad libitum. Animals (2–4 mo of age) were ovariectomized under Metofane (Janssen Pharmaceuticals, Ontario, Canada) anesthesia to remove ovarian steroid feedback and received steroid implants as described below. Postoperative analgesia was provided by a long-acting local anesthetic (bupivicaine 0.25%, 5 µl per site; Abbott Laboratories, North Chicago, IL). All procedures were approved by the Animal Care and Use Committee of the University of Virginia and conducted in accordance with the National Research Council publication Guide for Care and Use of Laboratory Animals.

Animal Models and Hormone Treatments

Experiments were done on acutely prepared brain slices from mice that had been ovariectomized and treated with hormone implants for 10 ± 2 days. This treatment duration was chosen because it is similar to the duration of the progesterone rise during pseudopregnancy in mice [43] and is also similar to the luteal phase rise in progesterone that occurs in species that do not exhibit the abbreviated reproductive cycle of small rodents. All mice received a Silastic (Dow Corning Co., Midland, MI) capsule containing 0.625 µg estradiol (OVX+E), as previously described [44]; this was used as the control condition, as E is required for progesterone receptor expression [45]. In addition to control mice, which received no further treatment, groups of mice were also treated with 1) a 2.5-mg progesterone time-release pellet (OVX+E+P; Innovative Research of America, Sarasota, FL), 2) a Silastic capsule containing 400 µg dihydrotestosterone (DHT) in sesame oil (OVX+E+DHT), or 3) P and DHT (OVX+E+P+DHT). DHT was chosen as the androgen treatment because it cannot be converted to estrogens. It thus acts exclusively at androgen receptors and provides a means to isolate androgen effects without confounds of additional estrogen actions. The DHT dose was chosen to approximate a high-normal testosterone (T) level in diestrous female mice (0.14 ± 0.03 ng/ml T, diestrus females, n = 13; 0.19 ± 0.04 ng/ml DHT, OVX+E+DHT, n = 9, P > 0.2). Because the androgen receptor has a somewhat higher affinity for DHT than for testosterone [46], the effective dose is higher than what a normal female would be exposed to, modeling the mild hyperandrogenemia associated with some clinical forms of hypothalamic infertility. The dose of progesterone used was chosen to achieve a P level similar to adult female mice from the same colony during estrus (1.7 ± 0.32 ng/ml, estrous females, n = 5; 1.8 ± 0.30 ng/ml, OVX+E+P, n = 6, P > 0.3). All hormones were administered in vivo and were not present in any recording solutions.

Endocrine status was confirmed on the day of the experiment by measuring serum progesterone and DHT in animals receiving those treatments (see above) and LH when remaining serum was available. LH levels were not different among control OVX+E, OVX+E+P, and OVX+E+P+DHT mice (0.11 ± 0.03 [SD] ng/ml, OVX+E, n = 7; 0.15 ± 0.04 ng/ml, OVX+E+P, n = 2; 0.14 ± 0.05 ng/ml, OVX+E+P+DHT, n = 3, P > 0.5). The lack of difference likely reflects all values being near the level of detection for this assay (range 0.07–0.11 ng/ml). In contrast, LH levels were elevated in OVX+E+DHT mice (0.29 ± 0.05 ng/ml, n = 8, P < 0.01 versus OVX+E), suggesting high-physiologic androgen levels upregulate reproductive neuroendocrine axis activity in female mice, as is seen in humans [7], and in contrast with the inhibition seen when intentionally supraphysiological levels of DHT have been administered [47].

Slice Preparation and Recordings

All reagents were purchased from Sigma Chemical Company (St. Louis, MO). Mice were killed between 0900 and 1100 h. Two hundred-micrometer coronal sections through the preoptic area and hypothalamus were prepared as previously described [48, 49]. The goal of this study was to compare GABAergic PSCs in GnRH neurons from the different animal models. By recording postsynaptic events using a high-chloride intracellular solution, with the membrane voltage clamped at –60 mV, and in the presence of glutamatergic receptor antagonists, GABAergic events are relatively isolated, the level of detection of these events is improved, and determination of the size of events is normalized between cells. Electrodes (2–4 M{Omega}) were filled with a high-chloride (140 mM) pipette solution with 4 mM MgATP and 0.4 mM NaGTP added before adjusting to pH 7.2 with NaOH [24, 50]. Details of recording parameters have been previously published [11, 24, 50]. To study the action-potential-independent effects of steroid treatments on GABAergic drive to GnRH neurons, tetrodotoxin (TTX, 0.5 µM) was applied to some cells from each treatment group after sufficient control data (minimum 3 x 2-min recording periods) had been obtained. In a subset of recordings in each group, elimination of all PSC activity after bath application of the GABAA receptor antagonist bicuculline at the end of the recording period (20 µM) confirmed PSC events were GABAA receptor-mediated. A maximum of one cell was recorded in each slice and up to five cells per animal.

PSC Analysis

Current traces were analyzed using custom detection software to identify PSCs (events) [24]. Cells were considered as independent observations, as within-cell variance was not different from within-group variance. Mean event frequency (in Hertz) from at least three 120-sec records was calculated for each cell to obtain mean PSC frequency for each cell; cells were then averaged to obtain group means. Group means were compared using one-way ANOVA, followed by post hoc analysis with Fisher protected least significant difference (LSD) test. Within a subset of individual cells in each group, frequency of GABAAR-mediated currents before and during in vitro treatment with tetrodotoxin (PSCs and mPSCs, respectively) was compared by paired Student-Newman-Keuls test when appropriate.

Averaged current waveforms were generated from all events in each 120-sec recording period for each cell after aligning events on the rising phase. These were used to illustrate differences in current amplitude. Averaged currents were then normalized by amplitude to show differences in decay time. Rate of rise (a measure of receptor on-rate), peak amplitude (a measure of conductance), and 10–90% decay time (a measure of off-rate/affinity) for every event detected were calculated by the program and analyzed in a spreadsheet (Microsoft Excel, Microsoft, Redmond, WA). To compare currents among in vivo treatment groups, group data sets were generated using 100 randomly selected events per cell or all events if fewer than 100 occurred [24]. These data are presented in probability plots, which show changes in the population of events at a greater level of detail than comparison of means [24, 51]. To generate these plots, all values for a given parameter in the group data set are sorted in ascending order and then normalized by the total number of events in that group. Normalization provides a rank distribution between 0 and 1 for every value; probability is plotted on the ordinate versus raw values on the abscissa. To compare currents recorded from a single cell before and after TTX treatment, probability distributions for interevent interval, rate of rise, amplitude, and decay time were generated during baseline (no TTX) and in vitro TTX treatment periods using all events recorded during the respective period. Probability distributions were compared between cells and within individual cells in different in vitro treatments, using the Kolmogorov-Smirnov goodness of fit test (SPLUS Professional 2 data analysis software; MathSoft, Inc., Cambridge, MA). For between-cell comparisons, mean values for rate of rise, amplitude, and decay time from each cell were then used to calculate mean percent change from control (OVX+E) in each in vivo treatment group. Means for each parameter were compared among treatment groups using one-way ANOVA followed by post hoc analysis with Fisher protected LSD and Student-Newman-Keuls for pairwise comparisons, when appropriate. All values are reported as mean ± SEM and significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Steroid Hormones Alter GABAergic Spontaneous PSC Frequency in GnRH Neurons

We first tested the hypothesis that steroid hormones alter GABAergic drive directly to GnRH neurons. To test this, we compared spontaneous PSC frequency in GnRH neurons from mice that were ovariectomized and treated with either estradiol alone (OVX+E, control), E plus progesterone (OVX+E+P), or E plus the nonaromatizable androgen DHT (OVX+E+DHT; Fig. 1). PSC frequency was significantly decreased and interevent interval increased in GnRH neurons from OVX+E+P compared with OVX+E control females (n = 6 mice, 24 cells, OVX+E; n = 6 mice, 24 cells, OVX+E+P, P < 0.04). In marked contrast, frequency was significantly increased and interevent interval reduced in the presence of DHT (n = 7 animals, 13 cells, OVX+E+DHT, P < 0.01). Bicuculline (20 µM) eliminated all events in all cells tested regardless of treatment group, demonstrating these currents were mediated via the GABAA receptor (Fig. 1A, bottom trace). These data suggest progesterone and androgen feedback independently alter presynaptic GABAergic drive to GnRH neurons.



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  		FIG. 1. Steroid hormones alter GABAergic PSC frequency in GnRH neurons. A) Recordings of GABAergic PSCs from a representative GnRH neuron from an ovariectomized, estradiol-treated female (OVX+E, control group) and from females that were also treated with either progesterone (OVX+E+P), the nonaromatizable androgen DHT (OVX+E+DHT), or both P and DHT (OVX+E+P+DHT) in vivo for 10 ± 2 days. These currents are mediated by the GABAAR, as illustrated by complete blockade by the GABAAR antagonist bicuculline (20 µM, OVX+E shown, bottom). B) Mean ± SEM spontaneous PSC frequency (Hz) in GnRH neurons from OVX+E (n = 24 cells), OVX+E+P (n = 24), OVX+E+DHT (n = 13), and OVX+E+P+DHT (n = 12) females. C) Probability distribution plot of interevent interval created using PSC events from all cells in each treatment group, show between-group comparisons for the interval between successive PSCs. *, P < 0.05 versus OVX+E control

Androgens may contribute to some forms of infertility by decreasing the efficacy of progesterone feedback [7, 9, 10]. To test if this interference might happen at the level of GABAergic drive, PSCs were recorded in mice treated with all three steroids (OVX+E+P+DHT). In these mice, PSC frequency and interevent interval were not different from OVX+E controls (n = 4 mice, 12 cells, P = 0.8, Fig. 1), suggesting progesterone and DHT interfere with one another at the level of output of the GABAergic afferent.

In general, changes in PSC frequency may be due to altered activity of presynaptic neurons, changes in the release probability of presynaptic vesicles, or an altered number of synaptic contacts between pre- and postsynaptic neurons. An initial test of which mechanisms were involved in this study is comparison of the frequency of GABAAR-mediated currents in a single GnRH neuron before and during in vitro treatment with TTX (0.5 µM). Currents recorded in the presence of TTX, which eliminates sodium-dependent action potentials, are known as miniature PSCs (mPSCs) and reflect the random, spontaneous release of single GABA vesicles from presynaptic cells rather than action-potential-driven release. Frequency of mPSCs has been shown to be directly proportional to the amount of physical synaptic contact [52]. Within-cell comparisons before and during TTX treatment indicate TTX had no effect on the frequency of GABAAR-mediated events in GnRH neurons from OVX+E+DHT-treated mice (1.8 ± 0.4 Hz pre, 1.7 ± 0.3 Hz during TTX, n = 6, P > 0.8). This suggests DHT may increase GABAergic drive to GnRH neurons by increasing the number of GABAergic contacts rather than by increasing the activity of GABAergic afferents. TTX similarly had no effect on frequency within cells in the other groups (OVX+E, 1.14 ± 0.3 Hz pre, 1.08 ± 0.3 Hz during TTX, n = 7, P > 0.7; OVX+E+P, 0.43 ± 0.2 Hz pre, 0.42 ± 0.1 Hz during TTX, n = 7, P > 0.8; OVX+E+P+DHT, 0.89 ± 0.3 Hz pre, 0.99 ± 0.4 Hz during TTX, n = 5, P > 0.4). Although perhaps indicative of changes in synaptic contacts induced by these various steroid regimens, these data are more difficult to evaluate in this regard, as any reductions in GABAergic drive due to reduced activity of presynaptic afferents would not be revealed by addition of TTX.

Steroid Hormones Alter Size Characteristics of GABAergic PSCs in GnRH Neurons

In addition to frequency, we also analyzed size characteristics of PSCs to determine if steroid treatments altered the response of GnRH neurons to GABAAR activation. Compared with OVX+E controls, PSC size was decreased in cells from OVX+E+P animals: specifically, rate of rise, a measure of receptor on-rate, and peak current amplitude, a measure of conductance, were both decreased in the presence of P (rate of rise, P < 0.01 versus OVX+E, amplitude, P < 0.04; Figs. 2 and 5A). Decay time (a measure of receptor off-rate) was not altered by P treatment (P = 0.4, Figs. 2 and 5A). Within-cell comparisons of PSCs and mPSCs (before and during in vitro treatment with TTX, respectively) in a subset of cells revealed the effect of P on size characteristics was not dependent on action-potential-induced activity, i.e., there was no further change in the presence of TTX (n = 7, P > 0.5 for rate of rise, amplitude, and decay time; Figs. 2B and 5B).



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  		FIG. 2. Progesterone diminished PSC size in GnRH neurons via activity-independent mechanism(s). A) Averaged (top) and normalized averaged (bottom) PSC traces from representative GnRH neurons from OVX+E and OVX+E+P mice show differences in spontaneous PSC amplitude and decay time, respectively, between the two groups. B) Probability distributions of PSC rate of rise (top), amplitude (middle), and decay time (bottom), created using PSC events from all cells in each treatment group, show between-group comparisons for these PSC properties and the lack of effect TTX has on these parameters. *, P < 0.05 versus OVX+E control



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  		FIG. 5. Summary of effects of progesterone, DHT, and the combination of these two steroids on PSC size in GnRH neurons. Key in (B) applies to the entire figure. A) Mean percent change from OVX+E control (n = 28 cells) in mPSC rate of rise, amplitude, and decay time summarize between-group comparisons in mPSC properties for all treatment groups (n = 18, OVX+E+P; n = 12, OVX+E+DHT; n = 13, OVX+E+P+DHT). *, P < 0.05 versus OVX+E control. B) Mean ± SEM percent change from baseline within individual GnRH neurons in each group in PSC rate of rise, amplitude, and decay time after treatment with TTX (n = 7, OVX+E+P; n = 6, OVX+E+DHT; n = 5, OVX+E+P+DHT). *, P < 0.05 versus baseline (pre-TTX) levels

In contrast with the inhibitory effects of progesterone on GABAergic PSCs, in OVX+E+DHT females, PSC rate of rise, amplitude, and decay time were all enhanced above control levels (P < 0.04 for each parameter versus OVX+E; Figs. 3 and 5A). Interestingly, TTX had marked effects on some measures of current size in DHT-treated animals. The mPSC rate of rise and amplitude in OVX+E+DHT mice were significantly (P < 0.03) less than OVX+E control values. Values for decay time, however, remained unchanged in the presence of TTX. That is, decay time in cells from OVX+E+DHT mice remained elevated relative to OVX+E controls during TTX treatment (P = 0.15; Figs. 3B and 5B). Effects of TTX on the rate of rise and amplitude of GABAAR-mediated currents suggest the increased response induced by DHT was largely due to increased activity-dependent presynaptic release of GABA. Further, the lack of a difference in decay time between PSCs and mPSCs indicates DHT may also act via multiple postsynaptic mechanisms to alter GnRH neuron responsiveness to GABAAR activation.



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  		FIG. 3. In vivo DHT increased GABAergic PSC size in GnRH neurons via activity-dependent mechanism(s). A) Averaged (top) and normalized averaged (bottom) spontaneous PSC traces from representative GnRH neurons from OVX+E and OVX+E+DHT mice show differences in PSC amplitude and decay time, respectively, between the two groups. B) Probability distributions of PSC rate of rise (top), amplitude (middle), and decay time (bottom), created using PSC events from all cells in each treatment group, show between-group comparisons for these PSC properties and the inhibitory effects of TTX on amplitude and rate of rise. *, P < 0.05 greater than OVX+E control; #, P < 0.05 less than OVX+E control

There were no differences in PSC rate of rise, amplitude, or decay time in GnRH neurons from OVX+E+P+DHT mice compared with OVX+E mice (P > 0.6 for each parameter, Figs. 4 and 5A). Within individual GnRH neurons from this group, the size of mPSCs was comparable with the size of PSCs (i.e., no parameter was altered by TTX, P > 0.5 for each parameter; Fig. 5B). Together with the frequency data (Fig. 1), these results suggest P and DHT may have counteracted each other's effects on the frequency and size characteristics of GABAAR-mediated currents in GnRH neurons.



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  		FIG. 4. PSC size is not different from controls in mice treated in vivo with both progesterone and DHT. Averaged (A) and normalized averaged (B) PSC traces from representative GnRH neurons from OVX+E and OVX+E+P+DHT mice show the similarity in PSC amplitude and decay time, respectively, in the two groups


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Steroid hormones are important regulators of GnRH release. The GABAergic system provides a major input to GnRH neurons [20–24] and has been proposed as a candidate mediator of steroid feedback [19, 27]. Here we demonstrate that in vivo treatment with progesterone or the nonaromatizable androgen DHT alters GABAergic drive directly to GnRH neurons. Progesterone, which, via negative feedback decreases pulsatile GnRH release, reduced both the frequency and size of GABAAR-mediated currents in GnRH neurons, whereas DHT had opposite effects. These data support and extend previous hypotheses on the importance of the GABAergic system in the regulation of GnRH neurons by directly demonstrating steroid-induced changes in GABAergic drive to these cells.

The changes in GABAergic PSC frequency following in vivo steroid treatment could arise from multiple mechanisms, including altered connectivity between GABAergic and GnRH neurons, changes in activity of presynaptic GABA neurons, and/or changes in the probability of vesicle release. The increase in PSC frequency in DHT-treated mice was not reversed by acute treatment of slices in vitro with TTX, which eliminates action-potential-dependent effects. This suggests an increase in the number of synaptic contacts from GABAergic neurons lead to the increase in frequency. Studies at the electron microscopic level will be required to confirm this hypothesis. Likewise, in progesterone-treated mice, frequency was similar before and after in vitro TTX treatment. In this case, however, PSC frequency was suppressed by progesterone. In vitro treatment with TTX can reveal activity-dependent increases in frequency but is not helpful in discerning if decreased frequency is due to reduced afferent activity or reduced connectivity. Both possibilities thus remain equally viable for progesterone action.

Steroid hormones have been shown to influence synaptic plasticity in GnRH and other neurons. Progesterone decreased the number of dendritic spiny projections, primary termination points for synaptic input, in hippocampal neurons [53]. In males, castration reduces spine formation in GnRH neurons [54]. Finally, GABAergic innervation of GnRH neurons in sheep is increased in the breeding season compared with anestrus, showing a positive correlation between GABAergic contacts and reproductive activity [20]. Together, these data suggest a relationship between the prevailing steroid hormone milieu and the degree of innervation GnRH neurons receive as a possible mechanism for the altered functional GABAergic drive observed in the present study.

There was no change in the frequency of GABAAR-mediated currents when action potentials were blocked. There are several possible explanations. First, in vivo steroid treatments were tested. Previous work indicates at least some steroid feedback effects persist in the slice preparation [44, 48], but some may not. Second, some afferents that convey steroid feedback will be removed by slice preparation. Activity-dependent effects were apparent, however, in the size of the currents, suggesting intact GABAergic afferents remain within the slice. Third, the changes may not be activity dependent but rather due to alternative mechanisms as proposed above. Finally, the standard protocols used to isolate GABAergic currents, specifically blockade of ionotropic glutamate currents, may alter the activity of presynaptic GABA neurons. In this regard, the same conditions were used in all groups, yet differences persisted. Preliminary observations in our lab suggest GABAergic PSC frequency in GnRH neurons is similar in the presence and absence of glutamatergic blockers (unpublished results). Further, changes in the frequency of GABAergic PSCs have been observed in other studies [23], confirming the cellular machinery for generating action potentials in these afferents is present within slice preparations.

In addition to altering the frequency of GABAergic PSCs, steroids also influenced the size of these currents. Such changes might reflect altered transmitter release from the presynaptic neuron, altered responsiveness of the postsynaptic neuron to GABAAR activation, or a combination of these. The progesterone-induced decrease in PSC size was not affected by TTX, suggesting this effect was activity independent. Presynaptically, progesterone may reduce the size, GABA concentration, or docking ability of vesicles. Postsynaptically, progesterone may alter GABAAR subunit expression [55–58], phosphorylation state [59], or GABA half-life in the synaptic cleft [60], all of which are known to regulate the amount of current that flows upon receptor activation [61]. It is also possible that progesterone acted directly on the GnRH neuron to alter the postsynaptic response to GABA. In this regard, molecular studies have thus far failed to show convincing evidence for nuclear progesterone receptor expression in GnRH neurons [17]. The possible role of a membrane-associated progesterone receptor [62] remains an interesting possibility to be examined in GnRH neurons.

In contrast with the reduction in PSC size induced by progesterone, DHT increased PSC size in GnRH neurons. Although the range of possible mechanisms mediating DHT and progesterone actions are similar, the different in vivo effects and responses to in vitro TTX treatment of each steroid suggest distinct mechanisms are involved. The DHT-induced increase in PSC rate of rise and amplitude were not only abolished by TTX but reduced below control values. This suggests DHT acts via distinct, opposing mechanisms: DHT stimulates GABA release from the presynaptic neuron, resulting in increased PSC size in the absence of TTX, yet inhibits the postsynaptic response to GABA, as revealed when presynaptic actions are minimized by TTX. Interestingly, TTX did not alter decay time in OVX+E+DHT mice, which remained elevated above control levels. DHT may thus act via a third, activity-independent mechanism to increase duration of GABAAR-mediated currents in GnRH neurons. Future studies will focus on parsing out the specific cellular and molecular mechanisms responsible for progesterone and DHT-induced changes in the response of GnRH neurons to GABAAR activation.

Progesterone and DHT had opposite effects on both the frequency of GABAergic drive to GnRH neurons and the magnitude of current that flows upon GABAA receptor activation. Increased frequency and size of GABA currents occurred in the DHT-treated animals, which also had elevated LH levels. This is consistent with several recent studies revealing a positive correlation between increased GABA action through the GABAAR on GnRH neurons and physiological measures of increased reproductive neuroendocrine activity [11, 23, 24]. The recording methods used in the present study were optimized for detection and quantitation of GABAergic currents and are not appropriate for concomitant examination of GnRH neuron function. Preliminary studies of long-term firing patterns of GnRH neurons using extracellular techniques indicate a reduced level of action-potential generation in GnRH neurons from progesterone-treated mice (unpublished results). This observation is also consistent with the hypothesis that the reduced GABA drive in such animals inhibits the firing activity likely associated with hormone release [63].

A recent report argues against this body of evidence, suggesting that the action of endogenous GABA on GnRH neurons is inhibitory [39]. In that study, neuronal network interactions were largely unaccounted for; thus, it is difficult to determine if the increased firing induced by GABAAR blockade was due to an inhibitory effect of GABA directly on GnRH neurons, as concluded by the authors, or due to the isolation of excitatory glutamatergic drive within the slice, which would be expected to increase activity [64]. Our own preliminary data suggest that, when GABAergic activity in the slice is isolated by blocking glutamatergic drive, subsequent blockade of GABAARs can result in an inhibition of firing rate in GnRH neurons [40].

Taken together with our previous observations of elevated chloride levels in GnRH neurons [37] and the positive correlation between activation of GABAARs in GnRH neurons and reproductive activity, we continue to favor the hypothesis that activation of GABAAR depolarizes these cells. When depolarization is large enough, action potentials and presumably hormone release will result. The steroid-induced changes in GABAAR-mediated currents observed in the present study will alter the probability that GABAAR activation depolarizes the cell to the threshold for action-potential firing. Depolarization is proportional to the size of the PSC. Further, the graded postsynaptic potentials that result from the flow of PSCs are additive; higher frequency PSCs increase the chance for temporal summation because there is a greater chance another PSC will arrive at the cell when the membrane is already depolarized. This can result in a greater overall depolarization and thus a greater chance for crossing the threshold for action-potential firing. Taken together, progesterone, which decreased the frequency and size of PSCs, would reduce the probability that presynaptic GABA release will trigger an action potential, whereas the probability for action-potential firing would be increased by DHT, which increased frequency and size of GABAergic inputs.

These changes are of interest to the neural regulation of reproduction because elevated androgens have been implicated in the pathogenesis of the common fertility disorder polycystic ovary syndrome (PCOS). Women with PCOS have high androgen levels and impaired follicular development due to consistently high frequency pulsatile LH (and presumably GnRH) release [7, 8]. One hypothesis that has emerged [7, 9–11] is that there is a hypothalamic origin for this disorder and that elevated androgens are causal. Specifically, high LH pulse frequencies result from GnRH neuron hyperactivity due to an androgen-mediated decrease in the sensitivity of these cells to progesterone negative feedback. Although the antagonism of progesterone negative feedback by androgens is of interest, it must be pointed out that the infrequency of ovulation in women with PCOS leads to a relative absence of progesterone. The role of elevated androgens in the function of the reproductive neuroendocrine axis without a concomitant presence of progesterone thus merits consideration.

The data presented here provide one possible central mechanism for the increased activity of this axis, i.e., that androgens contribute to excess GnRH neuron activity in PCOS by increasing GABAergic drive onto these cells and by increasing the response of GnRH neurons to GABAAR activation. Data consistent with this postulate include observations that androgens increase activity of GABAergic neurons in other brain regions [57, 65] and that central GABA levels are elevated in women with PCOS [66]. Recent work in prenatally androgenized mice as a model for PCOS revealed increased GABAergic drive that could be blocked by the androgen receptor antagonist flutamide [11]. In the present study, when mice were treated with both progesterone and DHT (OVX+E+P+DHT), GABAergic drive was similar to that seen in control animals treated only with estradiol (OVX+E), whereas drive in OVX+E+DHT animals was elevated above control levels. Together, these data suggest the GABAergic system is an important mediator of androgen interference with progesterone negative feedback to GnRH neurons and point to independent activating effects of androgens in this system.

The present study demonstrates that steroid feedback signals are both integrated by and communicated to GnRH neurons at least in part via the GABAergic system. In combination with previous work demonstrating GABAergic drive to GnRH neurons is altered by metabolic cues [23, 24] and the observation that GABAergic neurons provide a major input to GnRH neurons [20, 21], these data point to a working model in which GABAAR-mediated input to GnRH neurons is a primary mechanism for central regulation of fertility.


   ACKNOWLEDGMENTS
 
We thank Dr. Xu-Zhi Xu for expert technical assistance and Drs. Zhigou Chu, Cathy Christian, and Ilya Plonsky for editorial comments.


   FOOTNOTES
 
1 Supported by the National Institute of Child Health and Human Development/National Institutes of Health through cooperative agreement U54 HD28934, the U54 Ligand and Assay Core, and R01 HD41469. S.D.S. was supported by an individual Predoctoral National Research Service Award NS43871. Back

2 Correspondence: FAX: 434 982 0088; smm4n{at}virginia.edu Back

Received: 14 June 2004.

First decision: 2 July 2004.

Accepted: 17 August 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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