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Endocrinology Vol. 144, No. 10 4366-4375
Copyright © 2003 by The Endocrine Society

Neurosteroids Alter {gamma}-Aminobutyric Acid Postsynaptic Currents in Gonadotropin-Releasing Hormone Neurons: A Possible Mechanism for Direct Steroidal Control

Shannon D. Sullivan and Suzanne M. Moenter

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

Address all correspondence and requests for reprints to: Suzanne M. Moenter, Departments of Internal Medicine and Cell Biology, P. O. Box 800578, University of Virginia, Charlottesville, Virginia 22908. E-mail: smm4n{at}virginia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pulsatile GnRH release is required for fertility and is regulated by steroid feedback. Whether or not steroids or their metabolites act directly on GnRH neurons is not well established. In some neurons, steroid metabolites known as neurosteroids modulate the function of the GABAA receptor. Specifically, the progesterone derivative allopregnanolone is an allosteric agonist at this receptor, whereas the androgen dehydroepiandrosterone sulfate (DHEAS) is an allosteric antagonist. We hypothesized these metabolites act similarly on GnRH neurons to modify the response to GABA. Whole-cell voltage-clamp recordings of GABAergic miniature postsynaptic currents (mPSCs) were made from green fluorescent protein-identified GnRH neurons in brain slices from diestrous mice. Glutamatergic currents were blocked with antagonists and action potentials blocked with tetrodotoxin, minimizing presynaptic effects of treatments. Allopregnanolone (5 µM) increased mPSC rate of rise, amplitude and decay time by 15.9 ± 6.1%, 16.5 ± 6.3%, and 58.3 ± 18.6%, respectively (n = 7 cells). DHEAS (5 µM) reduced mPSC rate of rise (32.1 ± 5.7%) and amplitude (27.6 ± 4.3%) but did not alter decay time (n = 8). Effects of both neurosteroids were dose dependent between 0.1 and 10 µM. In addition to independent actions, DHEAS also reversed effects of allopregnanolone on rate of rise and amplitude so that these parameters were returned to pretreatment baseline values (n = 6). These data indicate allopregnanolone increases and DHEAS decreases responsiveness of GnRH neurons to activation of GABAA receptors by differentially modulating current flow through GABAA receptor chloride channels. This provides one mechanism for direct steroid feedback to GnRH neurons.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GNRH NEURONS ARE the central regulators of reproduction. Pulsatile release of GnRH from the hypothalamus is required for fertility (1) and drives the synthesis and secretion of pituitary gonadotropins, which act at the gonads to stimulate gametogenesis and steroidogenesis. Steroid hormones exert both positive and negative feedback (2, 3, 4) to regulate GnRH release across the reproductive cycle; however, the sites and mechanisms for this have not been fully elucidated. Steroids may affect GnRH output by altering synaptic drive to these cells, and/or by direct action on GnRH neurons. One mechanism for the latter may be activation of nuclear receptors recently described in GnRH neurons (5, 6, 7, 8), and/or membrane-associated steroid receptors implicated in the regulation of other neurons (9, 10).

Another possibility for direct action of steroids on GnRH neurons is as neurosteroids. Neurosteroids, synthesized from circulating precursors (11) or de novo within the central nervous system (12, 13), can modulate the response of a postsynaptic cell to {gamma}-aminobutyric acid (GABA) at the GABAA receptor (14, 15, 16, 17, 18). GABAergic neurons synapse on GnRH neurons (19, 20) and functionality of GABAA receptors has been demonstrated in these cells using electrophysiological approaches (21, 22, 23). GABAergic systems provide a major regulatory input to GnRH neurons (20, 24, 25, 26, 27, 28); thus, neurosteroid modulation of GABAA receptor function is of potential importance to feedback regulation to these cells.

The GABAA receptor is an intrinsic ion channel that passes mainly chloride (29). Depending on the combination of receptor subunits present (30, 31, 32, 33) and/or phosphorylation state (14, 34, 35), neurosteroids allosterically modulate the amount of current that flows when ligand binds this receptor, thereby altering the change in membrane potential induced by GABAA receptor activation (36). In neurosteroid-responsive cells, the progesterone derivative allopregnanolone is a potent enhancer of GABAA receptor function, increasing the response to GABA (36, 37, 38). The androgen derivative dehydroepiandrosterone-sulfate (DHEAS), in contrast, inhibits receptor function, decreasing the response to GABA (17, 18), and can abolish allopregnanolone-induced potentiation of GABAergic neurotransmission (39).

Both allopregnanolone (40) and DHEAS (41) can alter reproductive function when given in vivo, suggesting steroid feedback through this mechanism may play important physiological roles during the reproductive cycle and in cases of hypothalamic infertility. Whether these in vivo effects are due to neurosteroid modulation of GABAA receptor-mediated currents in GnRH or other neurons is an interesting question that has received little attention. Although a recent study indicated allopregnanolone can modulate the change in membrane potential induced by GABA (15), its effects on the underlying currents have not been studied. In addition, no information is available on the effects of DHEAS on these cells. In this study, we test the hypothesis that allopregnanolone and DHEAS modify the response of GnRH neurons to GABAA receptor activation, thus providing one candidate mechanism for direct steroid action on these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult female mice from an in-house, transgenic colony in which green fluorescent protein (GFP) is coexpressed with GnRH peptide (42) were used. Animals were housed in groups of three to five and maintained on standard rodent chow (Harlan 7012, Bartonsville, IL) and water ad libitum. All animals were held on a 14-h light, 10-h dark cycle with lights on at 0500 EST. Reproductive (estrous) cycle stage was determined by vaginal cytology. To control for cyclical changes in endogenous hormone levels, all experiments were done during diestrus. 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.

Slice preparation
All reagents were purchased from Sigma (St. Louis, MO). Brain slices were prepared as described previously (43). Briefly, the brain was rapidly removed and submerged in ice cold, oxygenated (95%O2-5%CO2) sucrose saline consisting of (in mM) 250 sucrose, 3.5 KCl, 26 NaHCO3, 10 glucose, 1.3 Na2HPO4, 1.2 MgSO4, and 2.5 MgCl2. Coronal slices (200 µm) through the preoptic area and hypothalamus were cut with a vibratome (Ted Pella, Inc., Redding, CA) and transferred to an oxygenated, 1:1 mixture of sucrose saline and recording solution, which contained (in mM) 130 NaCl, 3.5 KCl, 26 NaHCO3, 10 glucose, 1.3 Na2HPO4, 1.2 MgSO4, and 2.5 CaCl2, for 20 min at room temperature. Slices were then incubated in oxygenated recording solution at 30 C for at least 90 min before recording.

Recordings
For recording, individual slices were transferred to a recording chamber mounted on the stage of an Olympus BX50WI upright fluorescent microscope (Opelco, Dulles, VA). Slices in the recording chamber were continuously superfused at a rate of 5–6 ml/min with oxygenated recording saline kept at 30–32 C with an inline heating unit (Warner Instruments, Hamden, CT). All recordings were done in the presence of D(-)2-amino-5-phophonovaleric acid (20 µM) and 6-cyano-7-nitroquinoxaline-2,3-dione (10 µM) to block glutamatergic currents, and tetrodotoxin (TTX, 0.5 µM) to block action potentials. GFP-GnRH neurons were identified by brief (10–20 sec) illumination of fluorescence at 470 nm. Patch pipettes (2–5 M{Omega}) were filled with internal solutions as detailed below. A gigaseal (1–3 G{Omega}) was formed between the pipette and the cell membrane, and the whole-cell configuration was obtained. Series and input resistances were monitored regularly during recordings. Only recordings in which series resistance was less than 20 M{Omega} throughout the recording were included for analysis. Input resistances ranged from 500–900 M{Omega}. Experiments were performed using Pulse Control software (Instrutech, Port Washington, NY) and currents recorded with an EPC-8 amplifier (HEKA Electronics, Nova Scotia, Canada), digitized by an ITC-18 acquisition interface (Instrutech), and stored as Event Tracker files using IGOR PRO software (Wavemetrics, Lake Oswego, OR) on a G4 Macintosh computer (Apple Computer, Cupertino, CA).

Neurosteroids
Neurosteroid treatments included 3{alpha}-5{alpha}-tetrahydroprogesterone (allopregnanolone), 3ß-5{alpha}-tetrahydroprogesterone (3ß-isoform of allopregnanolone, inactive isomer), DHEAS, or allopregnanolone + DHEAS. Stock solutions of steroids were prepared in dimethylsulfoxide and diluted to 0.1% for recordings. This concentration of dimethylsulfoxide does not affect postsynaptic current (PSC) properties (43A ).

Dose-response studies
The dose response of GABAA receptor-mediated currents to neurosteroids was tested using brief, local application of GABA. Electrochemical driving force determined by the difference between holding potential and ECl (reversal potential for chloride currents, calculated by the Nernst equation) was adjusted by altering chloride levels in the pipette solution. This was done to control the magnitude of the response to GABA and optimize data analysis as detailed in the results. Electrodes were filled with a solution containing 60 mM Cl- to test the effects of allopregnanolone, or 80 mM Cl- to test the effects of DHEAS. The former consisted of, in mM, 60 KCl, 80 K gluconate, 10 HEPES, 5 EGTA, 4 MgATP, 0.4 NaGTP, 0.1 CaCl2 (pH 7.2); for the latter, KCl was adjusted to 80 mM and K gluconate to 60 mM. Local pressure application of GABA (0.1 mM in HEPES buffer, 5 msec, 5–10 {psi}) was accomplished as described previously (21). The GABAB receptor antagonist SCH50911 (10 µM) was included to eliminate responses to applied GABA via that receptor.

GABA-induced currents were detected in voltage-clamp at -60 mV with signals filtered at 7 kHz for several consecutive 120-second recording periods, between which series resistance, input resistance and cell capacitance were monitored (44). The initial 4–6 min (2–3 x 120-sec recording periods) were used to establish baseline values for each cell’s response to rapid GABA application in the bath solution. The next 4–6 min (2–3 x 120-sec periods) and every 4–6 min thereafter, cells were treated in vitro with increasing doses of either allopregnanolone or DHEAS in the recording solution, and the response to brief GABA application at each dose recorded. GABA was applied every 30–60 sec during each 120-sec recording period for a total of two to four applications per record; this intermittent application prevented rundown due to desensitization or disruption of the chloride gradient (21). Neurosteroid doses were 0.1, 0.5, 1.0, 2.0, 5.0, and 10 µM. Only cells in which a quality recording was maintained long enough to determine the response to all six doses were included for analysis. To verify that changes in the magnitude of response to rapid GABA were due to the neurosteroid rather than repeated activation of GABAA receptors, some cells were treated with higher doses first, followed by a 5- to 10-min washout period, and then treated with lower doses. Controls included stepping to the reversal potential for chloride (ECl) or addition of the GABAA receptor antagonist bicuculline (20 µM) to the bath solution; these demonstrated GABA-induced currents were 1) chloride-mediated and 2) specific to activation of GABAA receptors.

Recording miniature PSCs (mPSCs)
PSCs recorded in the presence of TTX are referred to as mPSCs and are due to random fusion of transmitter vesicles with the synaptic membrane rather than action potential-driven transmitter release. Because they occur in the absence of action potentials, mPSCs provide a direct measure of the postsynaptic effects of a drug with minimal influence from presynaptic actions. The traditional nomenclature for referring to postsynaptic currents generated by activation of GABAA receptors is inhibitory PSC (IPSC), whereas excitatory PSC (EPSC) refers to glutamatergic events. For GnRH neurons, however, this nomenclature is confusing, as adult GnRH neurons maintain high internal chloride (20–25 mM) and are thus excited by GABAA receptor activation (21). Thus, for GnRH neurons, instead of referring to these currents as IPSCs because they are GABAA receptor-mediated, or as EPSCs because they are excitatory, we refer to them as GABAergic PSCs, or simply PSCs. This avoids confusion between the function of GABAA receptors in GnRH neurons and the traditional nomenclature.

For recording mPSCs, patch electrodes were filled with a high chloride internal solution consisting of, in mM, 140 KCl, 10 HEPES, 5 EGTA, 4 MgATP, 0.4 NaGTP, 0.1 CaCl2 (pH 7.2); data were not corrected for the liquid junction potential of 3 mV (45). GFP-GnRH neurons were identified as described above, and the whole-cell configuration achieved. Voltage was clamped at -60 mV and signals filtered at 7 kHz for several consecutive 120-sec recording periods, between which series resistance, input resistance and cell capacitance were monitored. Similar recording conditions are typically used to optimize detection of endogenous GABAA receptor-mediated chloride currents (46), and result in an outward driving force on chloride ions such that chloride leaves the cell when these receptors are activated.

The initial 4–8 min (2–4 x 120-sec periods) of recording was used to establish baseline values for mPSC properties, after which neurosteroid treatment(s) were bath applied in vitro via the extracellular solution for 6–10 min (3–5 x 120-sec periods). In vitro neurosteroid treatments were followed by either treatment (6–10 min) with a second neurosteroid (for example, in vitro treatment with allopregnanolone followed by addition of DHEAS to the same extracellular solution) or washout (6–10 min) to examine reversibility. Neurosteroids were tested at 5 µM, which was near the maximal response observed in dose-response studies. Treatments included allopregnanolone, 3ß-allopregnanolone, an inactive isomer, DHEAS, or allopregnanolone + DHEAS. To ensure recorded mPSCs were mediated by the GABAA receptor, the GABAA receptor antagonist bicuculline (20 µM) was added to the extracellular solution in a subset of recordings; in all cases, bicuculline completely abolished mPSC activity.

Analysis of response to rapid GABA application
Large amplitude current spikes generated by brief, local application of GABA were analyzed manually using IGOR Pro software. The amplitude of each GABA-induced current event was measured as the distance from the recording baseline just before the GABA pulse to the peak current obtained. For each cell, the mean amplitude change was calculated for each dose and was normalized to the maximum mean amplitude for that particular cell. Because allopregnanolone enhances and DHEAS diminishes the response to GABA, the maximum amplitude in allopregnanolone-treated cells was the response obtained in 10 µM allopregnanolone, whereas the maximum amplitude in DHEAS-treated cells was the response obtained in the bath (untreated) solution. Dose response curves for allopregnanolone and DHEAS were then created by plotting neurosteroid concentration [Log (µM)] vs. the mean normalized amplitude for all cells at that dose.

mPSC analysis
Stored 120-sec records of current activity were analyzed off-line using custom event detection tools designed with IGOR PRO software to identify mPSCs (events). Threshold for event detection was set manually for each 120-sec record and was typically at least 5 pA. Events were confirmed by eye and detection errors were corrected manually to ensure no events were left undetected. This allowed for accurate measurement of mPSC frequency as well as accurate assessment of neurosteroid effects on mPSC amplitude. Only cells in which a total of >=50 events were detected were included for analysis (mean event frequency for all cells was 0.40 ± 0.06 Hz). Mean event frequency from 2–4 x 120-sec records was calculated for each cell during baseline and treatment periods to obtain mean mPSC frequency before and after treatment. Rate of rise (a measure of receptor on-rate), peak amplitude (a measure of conductance), and decay time (a measure of off-rate/affinity) for every event detected were calculated by the program and exported for further analysis in a spreadsheet (Microsoft Excel, Microsoft, Redmond, WA). For each cell, cumulative probability plots for each parameter were generated during baseline and treatment periods using all events recorded during the respective periods. To do this, values for a given mPSC parameter during each treatment period were sorted in ascending order, and then normalized by the total number of events during that period. Normalization provided a probability rank distribution between 0 and 1 for every raw value; this probability was plotted on the y-axis vs. the raw values on the x-axis (47). Probability distributions among within-cell treatments were compared with the Komolgorov-Smirov Goodness of Fit test (SPLUS Professional 2 data analysis software, MathSoft, Inc., Cambridge, MA). Mean percent change from baseline for each treatment was then calculated for each parameter. Means for each mPSC parameter were compared among treatment groups using one-way ANOVA followed by post hoc analysis with Fisher’s protected least significant difference and Student-Newman-Keuls for pair-wise comparisons when appropriate. For each 120-sec recording period, an average mPSC waveform was generated from all mPSC events detected. For each cell, averaged mPSC waveforms were aligned on the rising phase for comparison between baseline and treatment conditions. These averaged mPSCs were used to illustrate changes in mPSC amplitude before and after treatment(s). The averaged mPSC waveforms were then normalized by amplitude to illustrate differences in mPSC decay time between treatments. 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
 
Effects of allopregnanolone on the response to brief, local application of GABA
To determine whether GABAA receptors expressed by GnRH neurons are sensitive to allosteric modulation by allopregnanolone, we recorded the response of these cells to brief, local pressure application of GABA (0.l mM) in the presence of various doses of allopregnanolone (0.1, 0.5, 1, 2, 5, and 10 µM). For recording the effects of allopregnanolone on GABA-evoked currents, we used an intracellular recording solution containing 60 mM chloride (vs. 140 mM extracellular chloride) to create a submaximal electrochemical driving force on chloride (~38 mV at -60 mV holding potential). This ensured that GABA-induced currents remained less than 1000 pA in amplitude at all allopregnanolone doses tested, thus allowing for more accurate measurement of amplitude changes by the acquisition software.

At all doses tested, allopregnanolone increased the amplitude of currents induced by GABA application. The response of an individual GnRH neuron to GABA in the presence of successively increasing doses of allopregnanolone is shown in Fig. 1AGo. Maximal effects on current amplitude occurred at 5–10 µM allopregnanlone, with an EC50 of 1 µM (n = 4, Fig. 1BGo). In the presence of the highest dose of allopregnanolone tested (10 µM), current induction by GABA was completely eliminated by holding cells at the reversal potential for chloride (ECl = -22 mV), indicating these currents were chloride-mediated (Fig. 1CGo). Further, specificity of GABA-induced current induction for activation of GABAA receptors was demonstrated by blockade with the GABAA receptor antagonist bicuculline (20 µM, Fig. 1CGo).



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  		FIG. 1. Allopregnanolone increases current flow through the GABAA receptors of GnRH neurons in a dose-dependent manner. Panel A, Representative examples from a single cell of current induced by a brief, local puff of GABA (0.1 mM) at each dose of allopregnanolone (0.1, 0.5, 1, 2, 5, and 10 µM). Panel B, Dose-response curve for allopregnanolone in GnRH neurons; C, untreated control. Panel C, Holding cells at the reversal potential for chloride (-22 mV) or addition of the GABAA receptor antagonist bicuculline to the bath solution eliminates current in response to a local GABA puff in the presence of allopregnanolone.

 
Effects of allopregnanolone on GABAergic mPSC properties
Because allopregnanolone enhanced GABA-evoked currents in GnRH neurons, we next asked if endogenous GABAA receptor-mediated currents are sensitive to allosteric modulation by allopregnanolone in these cells. To test this we recorded mPSCs before and after addition of allopregnanolone (5 µM) to the extracellular solution. mPSCs are due to random fusion of transmitter vesicles with the synaptic membrane, and are thus typically much smaller than currents induced by exogenous GABA. To increase our ability to determine any effects of allopregnanolone on this endogenous activity, we made two modifications from the previous experiment. First, we used an allopregnanolone dose near the top of the dose-response curve (5 µM, Fig. 1Go). Second, we altered recording conditions to increase the electrochemical driving force on chloride currents and thus enhance detection. Specifically, for these and other experiments testing neurosteroid effects on endogenous mPSCs, a high chloride (140 mM, equal to extracellular chloride levels) intracellular recording solution was used and membrane potential held at -60 mV. Under these conditions, the electrochemical driving force on chloride is approximately 60 mV.

We examined changes in mPSC frequency, rate of rise (a measure of receptor on-rate), amplitude (a measure of conductance), and decay time (a measure of off-rate/affinity). As expected in the presence of TTX to block action potentials, allopregnanolone had no effect on mPSC frequency (n = 7, baseline 0.34 ± 0.11 Hz, allopregnanolone 0.30 ± 0.11 Hz, P > 0.8), suggesting minimal presynaptic influences were present and that postsynaptic actions of treatments predominated. Allopregnanolone increased mPSC rate of rise, amplitude, and decay time (n = 7, P < 0.04). To illustrate changes in mPSC amplitude and decay time induced by allopregnanolone, mPSCs recorded before and after treatment were averaged to show amplitude changes and then normalized by amplitude to show changes in decay time. Representative averaged and normalized averaged mPSCs (Fig. 2AGo) show 5 µM allopregnanolone increased both mPSC amplitude and decay time. In our observations of GnRH neurons, mPSC properties often vary substantially from cell to cell, perhaps due to differences in the amount or type of GABAA receptors expressed by the cells (48). To demonstrate this variability, and to illustrate allopregnanolone-induced increases in mPSC rate of rise, amplitude, and decay time for individual cells, raw values and direction of change are plotted for each property examined for five representative cells from this treatment group (Fig. 2BGo). Elimination of all mPSC activity by the GABAA receptor antagonist bicuculline (20 µM) confirmed these currents were mediated specifically by GABAA receptors (Fig. 2CGo). In control experiments, the response of GnRH neurons to the inactive isoform of allopregnanolone, 3ß-5{alpha}-tetrahydroprogesterone (3ß-isoform, 5 µM), was tested. The 3ß-isoform of allopregnanolone had no effect on mPSC rate of rise (P > 0.3), amplitude (P > 0.4), or decay time (P > 0.1, n = 6, Fig. 2DGo), indicating the response to the active 3{alpha}-isoform was specific to this neurosteroid and not secondary to changes in membrane fluidity induced by steroid intercolation, or to vehicle. Cumulative results of this study are summarized in Fig. 2DGo, which plots the mean (±SEM) percentage change from baseline for mPSC rate of rise, amplitude, and decay time after treatment with 5 µM of either the active 3{alpha}-isoform (filled bars) or the inactive 3ß-isoform of allopregnanolone (unfilled bars). Together, these data indicate allopregnanolone directly increased current flow through GABAA receptors in postsynaptic GnRH neurons without changing the frequency of presynaptic GABAergic drive.



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  		FIG. 2. Allopregnanolone (5 µM) affects multiple properties of endogenous mPSCs. A, Averaged (left) and normalized average (right) mPSCs from an individual GnRH neuron before and after treatment with allopregnanolone. B, Raw data from five representative cells showing effects of allopregnanolone on mPSC rate of rise (left), amplitude (center) and decay time (right). Arrows indicate direction of response upon treatment with allopregnanolone. C, Representative recording trace shows bicuculline blocks all mPSCs (downward deflections) in the presence of allopregnanolone, demonstrating these currents are mediated by activation of the GABAA receptor. D, Mean (±SEM) percent change from baseline in mPSC rate of rise, amplitude, and decay time after treatment with allopregnanolone (black bars) or the inactive 3ß-isoform of allopregnanolone (3ß-5{alpha}-tetrahydroprogesterone, white bars). *, P < 0.05.

 
Effects of DHEAS on the response to brief, pressure application of GABA
To determine if GABAA receptors expressed by GnRH neurons are sensitive to allosteric modulation by DHEAS, we recorded the response of GnRH neurons to brief, local pressure application of GABA (0.l mM) in the presence of various doses of DHEAS (0.1, 0.5, 1, 2, 5, and 10 µM). For recording GABA-evoked currents in the presence of DHEAS, we used an intracellular recording solution containing 80 mM chloride while holding cells at -60 mV, producing a driving force of approximately 45 mV. Similar to the above experiments using allopregnanolone, these recording conditions favored outward chloride currents under the influence of relatively low electrochemical driving force to ensure GABA-induced currents remained less than 1000 pA in peak amplitude. In these experiments, however, a slightly higher chloride concentration was used in the pipette solution, as DHEAS is an allosteric antagonist at GABAA receptors and was hypothesized to decrease rather than increase the size of GABA-induced currents. Increasing intracellular chloride increased the driving force on chloride, allowing more accurate measurement of decreases in current size from baseline.

At the doses tested, DHEAS decreased the size of large amplitude currents induced by local GABA application. The response of an individual GnRH neuron to GABA in the presence of successively increasing doses of DHEAS is shown in Fig. 3AGo. Maximal effects on current amplitude occurred at 5–10 µM, with an EC50 of 0.8 µM (n = 2, Fig. 3BGo). At the highest dose of DHEAS tested (10 µM), current induction by GABA was completely eliminated by holding cells at the reversal potential for chloride (ECl = -15 mV), verifying these were chloride currents (Fig. 3CGo). Further, evidence these currents were specifically mediated by GABAA receptors was demonstrated by similar blockade with the GABAA receptor antagonist bicuculline (20 µM, Fig. 3CGo).



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  		FIG. 3. DHEAS decreases current flow through the GABAA receptor in a dose-dependent manner. Panel A, Representative examples from a single cell of current induced by a brief, local puff of GABA (0.1 mM) at each dose of DHEAS (0.1, 0.5, 1, 2, 5, and 10 µM). Panel B, Dose-response curve for DHEAS in GnRH neurons; C, untreated control. Panel C, Holding cells at the reversal potential for chloride (-22 mV) or addition of the GABAA receptor antagonist bicuculline to the bath solution eliminates current in response to a local GABA puff in the presence of DHEAS.

 
Effects of DHEAS on GABAergic mPSC properties
We next recorded mPSCs from GnRH neurons before and after addition of the androgen derivative DHEAS (5 µM) to the extracellular solution. Because mPSCs are much smaller than currents induced by exogenous GABA, we chose a dose of DHEAS near the top of the dose-response curve (5 µM, Fig. 3Go), and recording conditions creating relatively strong outward drive on chloride-mediated currents as described previously. As expected in the presence of TTX to minimize presynaptic effects, DHEAS did not alter mPSC frequency (0.66 ± 0.20 Hz baseline, 0.64 ± 0.17 Hz DHEAS, P > 0.5). DHEAS had a significant inhibitory effect on current rate of rise (P < 0.01) and amplitude (P < 0.01) but did not alter decay time (P > 0.1, n = 8, Fig. 4AGo, representative averaged and normalized averaged mPSCs from an individual cell). To illustrate between-cell variability in baseline mPSC properties, raw values and direction of change in response to DHEAS are plotted for mPSC rate of rise, amplitude, and decay time for five representative cells from this treatment group (Fig. 4BGo). These data are summarized in Fig. 4CGo, which plots the mean (±SEM) percentage change from baseline for mPSC rate of rise, amplitude, and decay time after treatment with 5 µM DHEAS. In control experiments, all mPSC activity in the presence of DHEAS was eliminated by the GABAA receptor antagonist bicuculline (20 µM, not shown), evidence these currents are GABAA receptor-mediated. Taken together, these data indicate DHEAS decreased current flow through GABAA receptors in GnRH neurons, thus reducing the response of these cells to GABA. Further, the lack of change in mPSC frequency despite the reduced size of these events in the presence of DHEAS indicates the ability to detect small events did not limit analyses; i.e. there was no reduction in frequency secondary to impaired detection of events that were reduced in size by treatment.



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  		FIG. 4. DHEAS (5 µM) affects multiple properties of endogenous mPSCs. A, Averaged (top) and normalized average (bottom) mPSCs from individual GnRH neurons before and after treatment with DHEAS. B, Raw data from five representative cells showing effects of DHEAS on mPSC rate of rise (left), amplitude (center) and decay time (right). Arrows indicate direction of response upon treatment with DHEAS. C, Mean (±SEM) percent change from baseline in mPSC rate of rise, amplitude, and decay time after treatment with DHEAS. *, P < 0.05.

 
Does DHEAS reverse effects of allopregnanolone on mPSC properties?
DHEAS has been shown to reverse the stimulatory effects of allopregnanolone on GABAA receptor function (39); thus we next tested whether or not DHEAS alters the response of GnRH neurons to allopregnanolone. We chose to look at endogenous GABAergic activity, i.e. mPSCs, for this study, as effects on endogenous currents would have greater physiological significance than effects on induced currents. Thus, we next recorded mPSCs before and after in vitro addition of allopregnanolone (5 µM) to the extracellular solution, and then after subsequent addition of DHEAS (5 µM). DHEAS reversed effects of allopregnanolone on mPSC rate of rise and amplitude but had no effect on decay time in the presence of allopregnanolone. Current traces from a single GnRH neuron before and during treatment with allopregnanolone, and then after subsequent addition of DHEAS, are shown in Fig. 5Go. Averaged and normalized averaged mPSCs from this cell (Fig. 6AGo) more clearly depict increases in current amplitude (top) and decay time (bottom) in the presence of allopregnanolone, as well as the reversal of this allopregnanolone effect on amplitude but not decay time after addition of DHEAS. The inability of DHEAS to alter mPSC decay time in the presence of allopregnanolone was not surprising, as this androgen similarly had no effect on decay time when given alone. Cumulative probability distributions of mPSC rate of rise, amplitude, and decay time, created from all mPSCs recorded during each treatment period from a single GnRH neuron (representative cell in Fig. 6CGo), clearly illustrate allopregnanolone increased (right shift) all three parameters of postsynaptic response as reported above. Subsequent treatment with DHEAS reversed effects of allopregnanolone on rate of rise and amplitude, indicated by left shifts in the respective probability distributions for DHEAS plus allopregnanolone vs. allopregnanolone alone (Fig. 6CGo, top and center, P > 0.05 vs. pretreatment baseline values). In contrast, DHEAS had no effect on decay time in the presence of allopregnanolone (Fig. 6CGo, bottom, P < 0.05 vs. baseline, P > 0.05 vs. allopregnanolone alone, no shift in the decay time distribution plot after addition of DHEAS). Data from all cells in this group (n = 6) are summarized as mean (±SEM) percentage change from baseline for mPSC rate of rise, amplitude, and decay time after simultaneous treatment with both 5 µM allopregnanolone and 5 µM DHEAS (Fig. 6BGo). DHEAS reversed effects of allopregnanolone on mPSC rate of rise and amplitude such that percent changes from pretreatment baseline values were no longer statistically significant but did not alter mPSC decay time. In summary, the data presented here indicate GABAA receptor function in GnRH neurons can be directly modulated by allopregnanolone and DHEAS, providing one mechanism for direct steroid feedback to these cells.



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  		FIG. 5. Representative current traces from a single GnRH neuron during baseline recording (top), allopregnanolone treatment (5 µM, center), and subsequent addition of DHEAS (5 µM, bottom). mPSCs are the downward deflections. Dotted lines in center and bottom traces indicate mean control amplitude.

 


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  		FIG. 6. DHEAS reverses the effects of allopregnanolone on some, but not all, mPSC properties. A, Averaged (top) and normalized average (bottom) mPSCs from an individual GnRH neuron show allopregnanolone increased mPSC amplitude and decay time, respectively, and that DHEAS reversed effects on amplitude only. B, Mean (±SEM) percent change from baseline in mPSC rate of rise, amplitude, and decay time after simultaneous treatment with both allopregnanolone and DHEAS. C, Cumulative probability distributions from representative individual GnRH neurons show allopregnanolone increased mPSC rate of rise (top), amplitude (center), and decay time (bottom), and DHEAS reversed these effects on rate of rise and amplitude, but not decay time. *, P < 0.05 vs. bath.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Actions of GABAergic networks through the GABAA receptor provide a major source for regulating GnRH neurons (20, 21, 23, 24, 25, 26, 27, 28). The ability of steroid hormones or their metabolites to modify these inputs is thus of potential importance in fertility regulation. The neural progesterone derivative allopregnanolone and the androgen derivative DHEAS are known allosteric modulators at the GABAA receptor, providing one candidate mechanism for direct steroid feedback to GnRH neurons. The sensitivity of a cell to allosteric regulation by a neurosteroid depends on both the combination of GABAA receptor subunits present (30, 31, 32, 33) and receptor phosphorylation state (14, 34, 35); thus, not all cells expressing GABAA receptors respond to all allosteric modulators. In the present study, we tested whether or not GnRH neurons are sensitive to direct neurosteroid regulation of the postsynaptic response to GABAA receptor activation. Effects of allopregnanolone and DHEAS presented here indicate GABAA receptors of GnRH neurons in diestrous female mice are sensitive to both of these neurosteroids.

The physiological consequence of GABAA receptor activation, and thus the result of neurosteroid action, depends on both the membrane potential and the intracellular chloride ion concentration of a cell. Previous work suggested GABAA receptor agonists decrease and GABAA receptor antagonists increase GnRH release (49, 50); however, those studies could not ascertain whether effects occurred at the GnRH neuron itself and/or at presynaptic cells. Recently, adult GnRH neurons were shown to maintain high intracellular chloride levels such that the chloride reversal potential is depolarized relative to the threshold for action potential firing. Direct activation of GABAA receptors on these cells is thus excitatory (21), suggesting increased GnRH release in previous studies using bath or intraventricular administration of GABAA receptor antagonists (49) was due to presynaptic actions.

The results of the present study indicate allopregnanolone and DHEAS, at doses similarly effective in other neural phenotypes (38, 39, 51, 52), can alter the magnitude of this excitatory response via direct allosteric action at GnRH neuron GABAA receptors. This alters receptor affinity for ligand and thus the probability receptors will be in the open state. Specifically, this study shows allopregnanolone increased, whereas DHEAS decreased, the current generated in GnRH neurons by GABAA receptor activation, and thus the change in membrane potential elicited when GABAA receptors are activated. These changes should enhance and diminish, respectively, the efficacy of excitatory GABAergic drive the GnRH neuron receives, and thus the probability for action potential firing and ultimately, for hormone secretion. In that regard, allopregnanolone has been shown to induce GnRH release from immortalized GnRH (GT1) neurons via direct action on GABAA receptors (53). Furthermore, it is important to note that the net effect of these neurosteroids on in vivo GnRH neuron output may depend on their ability to interact not only with GABAergic, but also with other neurotransmitter systems. For example, DHEAS decreases N-methyl-D-aspartic acid type glutamate receptor-mediated calcium currents in the hippocampus, resulting in reduced neuronal excitability (54, 55). Similar actions of DHEAS at GnRH neuron N-methyl-D-aspartic acid type glutamate receptors would suggest inhibitory signals from this neurosteroid are communicated to the reproductive axis via multiple, possibly additive pathways.

These results support a previous study in GnRH neurons in which allopregnanolone enhanced changes in membrane potential induced by GABA (15). The present study extends those findings to include effects of the androgen derivative DHEAS, interactions between DHEAS and allopregnanolone, as well as a direct examination of the currents produced by GABAA receptor activation. Importantly, the voltage-clamp approach used in the present study eliminates possible activation of voltage-dependent channels in GnRH neurons (44, 56, 57, 58), which could have contributed to the previous results (15). Together with the previous study, these data indicate the GABAA receptors of adult GnRH neurons are susceptible to allosteric modulation by both allopregnanolone and DHEAS, providing a possible mechanism for direct steroid feedback to these cells.

In that regard, neurosteroids are positioned to play an important role in the central control of GnRH output. Sensitivity of GABAA receptors to neurosteroids varies with the particular combination of receptor subunits present (30, 31, 32, 33) as well as receptor phosphorylation state (14, 34, 35). Hypothalamic expression of various GABAA receptor subunits (59, 60, 61), central neurosteroid levels (40, 62, 63), and sensitivity to neurosteroids (59, 61) have all been shown to vary across the reproductive cycle and in different reproductive states. In GnRH neurons, mechanisms including altered neurosteroid sensitivity secondary to changes in GABAA receptor subunit expression and/or phosphorylation state, and fluctuations in neurosteroid levels could contribute to the multifactorial control of this important neuroendocrine system. This may have implications for the regulation of GnRH neuron output during both the normal reproductive cycle and in various states of infertility.

With regard to fertility regulation, the allopregnanolone-induced increase in GnRH neuron responsiveness to GABAA receptor activation is interesting because it indicates allopregnanolone potentiated an excitatory signal to GnRH neurons, whereas its precursor progesterone is known to provide potent negative feedback to these cells (2, 4, 64). For example, preliminary evidence indicates progesterone diminishes excitatory GABAergic drive to GnRH neurons by decreasing both the frequency and size of GABAA receptor-mediated currents (65). Whether the excitatory effect of allopregnanolone or the inhibitory effect of progesterone predominates over in vivo GnRH neuron activity would thus depend on relative brain levels of the two steroids as well as sensitivity to these factors.

This is of interest with regard to both normal reproduction and a common form of hypothalamic infertility, polycystic ovary syndrome (PCOS), marked by increased LH (and presumably GnRH) pulse frequency (66) and elevated androgen levels (67). Androgens up-regulate the synthetic enzymes for allopregnanolone, 5-{alpha}-reductase and 3-{alpha}-hydroxysteroid dehydrogenase (68). In females with normal androgen levels, the conversion to allopregnanolone would thus be expected to be lower than in hyperandrogenic females. In this regard, androgen excess has been proposed to contribute to the reduced efficacy of progesterone negative feedback and subsequent elevation in LH pulse frequency characteristic of PCOS (69, 70, 71). Of interest, it was recently reported that brain GABA levels are also elevated in PCOS (72). It is possible that elevated levels of both GABA and androgen contribute to GnRH neuron hyperactivity by increasing GABAergic tone and conversion to allopregnanolone, respectively, thus shifting the feedback balance toward the excitatory neurosteroid. A possible caveat to this hypothesis is that DHEAS, which decreases responsiveness of GnRH neurons to GABAA receptor activation, is also elevated in women with PCOS (67). The data presented here, however, indicate allopregnanolone has a more pronounced effect on GABAA receptor-mediated currents in GnRH neurons; that is, DHEAS did not fully suppress the stimulatory effects of allopregnanolone at GnRH neuron GABAA receptors. Of course, this does not eliminate the possibility that DHEAS may also exert important inhibitory actions via the glutamatergic system (54, 55). Future work is needed to examine the precise mechanisms by which these neurosteroids may interact to alter GnRH neuron excitability in fertile and hyperandrogenic states.

Together, these data indicate the neurosteroids allopregnanolone and DHEAS modify the response of GnRH neurons to GABAA receptor activation, providing a potential mechanism for direct regulation of GnRH neurons by steroid hormones. A majority of the functional synaptic input to GnRH neurons demonstrated to date arises from GABAergic inputs (20, 21, 22, 23); thus, neurosteroids are poised to exert significant effects on GnRH neuron activity. They may play important roles in regulating the normal reproductive cycle as well as contribute to various forms of hypothalamic infertility. Future studies will address the net effect of direct and indirect feedback of steroids and their derivatives on GnRH neuron activity and ultimately fertility.


   Footnotes
 
We thank Xu-Zhi Xu for excellent technical support.

This work was supported by National Institute of Child Health and Human Development/NIH through cooperative agreement U54 HD28934 as part of the Specialized Cooperative Centers Program in Reproduction Research. S.D.S. was supported by F31 NS43871.

Abbreviations: DHEAS, Dehydroepiandrosterone sulfate; EPSC, excitatory PSC; GABA, {gamma}-aminobutyric acid; GABAA, A subtype of GABA receptor; GFP, green fluorescent protein; IPSC, inhibitory PSC; mPSC, miniature PSC; PCOS, polycystic ovary syndrome; PSC, postsynaptic current; TTX, tetrodotoxin.

Received May 23, 2003.

Accepted for publication July 8, 2003.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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