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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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Belchetz P, Plant TM, Nakai Y, Keogh EJ, Knobil E 1978 Hypophysial responses to continuous and intermittent delivery of hypothalamic goadotropin-releasing hormone. Science 202:631–633[Abstract/Free Full Text]
  2. Leipheimer RE, Bona-Gallo A, Gallo RV 1984 The influence of progesterone and estradiol on the acute changes in pulsatile luteininzing hormone release induced by ovariectomy on diestrus day 1 in the rat. Endocrinology 114:1605–1612[Abstract/Free Full Text]
  3. Moenter SM, Caraty A, Locatelli A, Karsch FJ 1991 Pattern of GnRH secretion leading up to ovulation in the ewe: existence of a preovulatory GnRH surge. Endocrinology 129:1175–1182[Abstract/Free Full Text]
  4. Kasa-Vubu JZ, Dahl GE, Evans NP, Thrun LA, Moenter SM, Padmanabhan V, Karsch FJ 1992 Progesterone blocks the estradiol-induced gonadotropin discharge in the ewe by inhibiting the surge of gonadotropin-releasing hormone. Endocrinology 131:208–212[Abstract/Free Full Text]
  5. Herbison AE, Pape JR 2001 New evidence for estrogen receptors in gonadotropin-releasing hormone neurons. Front Neuroendocrinol 22:292–308[CrossRef][Medline]
  6. Hrabovszky E, Steinhauser A, Barabas K, Shughrue PJ, Petersen SL, Merchenthaler I, Liposits Z 2001 Estrogen receptor-ß immunoreactivity in luteinizing hormone-releasing hormone neurons of the rat brain. Endocrinology 142:4094–4095[Abstract/Free Full Text]
  7. Hrabovszky E, Shughrue PJ, Merchenthaler I, Hajszan T, Carpenter CD, Liposits Z, Petersen SL 2000 Detection of estrogen receptor-ß messenger ribonucleic acid and 125I-estrogen binding sites in luteinizing hormone-releasing hormone neurons of the rat brain. Endocrinology 141:3506–3509[Abstract/Free Full Text]
  8. Butler JA, Sjoberg M, Coen CW 1999 Evidence for oestrogen receptor {alpha}-immunoreactivity in gonadotrophin-releasing hormone-expressing neurones. J Neuroendocrinol 11:331–335[CrossRef][Medline]
  9. Zhu Y, Bond J, Thomas P 2003 Identification, classification, and partial characterization of genes in humans and other vertebrates homologous to a fish membrane progestin receptor. Proc Natl Acad Sci USA 100:2237–2242[Abstract/Free Full Text]
  10. Toran-Allerand CD, Guan X, MacLusky NJ, Horvath TL, Diano S, Singh M, Connolly ESJ, Nethrapalli IS, Tinnikov AA 2002 ER-X: a novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury. J Neuroscience 22:8391–8401[Abstract/Free Full Text]
  11. Barnea A, Hajibeigi A, Trant JM, Mason JI 1990 Expression of steroid metabolizing enzymes by aggregating fetal brain cells in culture: a model for developmental regulation of the progesterone 5 {alpha}-reductase pathway. Endocrinology 127:500–502[Abstract/Free Full Text]
  12. Jung-Testas, I Do Thi A, H K, Desarnaud F, Shazand K, Schumacher M, Baulieu EE 1999 Progesterone as a neurosteroid: synthesis and actions in rat glial cells. J Steroid Biochem Mol Biol 69:97–107[CrossRef][Medline]
  13. King SR, Manna PR, Ishii T, Syapin PJ, Ginsberg SD, Wilson K, Walsh LP, Parker KL, Stocco DM, Smith RG, Lamb DJ 2002 An essential component in steroid synthesis, the steroidogenic acute regulatory protein, is expressed in discrete regions of the brain. J Neurosci 22:10613–10620[Abstract/Free Full Text]
  14. Fancsik A, Linn DM, Tasker JG 2000 Neurosteroid modulation of GABA IPSCs is phosphorylation dependent. J Neuroscience 20:3067–3075[Abstract/Free Full Text]
  15. Sim JA, Skynner MJ, Herbison AE 2001 Direct regulation of postnatal GnRH neurons by the progesterone derivative allopregnanolone in the mouse. Endocrinology 142:4448–4453[Abstract/Free Full Text]
  16. Mtchedlishvili Z, Bertram EH, Kapur J 2001 Diminished allopregnanolone enhancement of GABAA receptor currents in a rat model of chronic temporal lobe epilepsy. J Physiol 537:453–465[Abstract/Free Full Text]
  17. Majewska MD, Demirgoren S, Spivak CE, London ED 1990 The neurosteroid dehydroepiandrosterone sulfate is an allosteric antagonist of the GABAA receptor. Brain Res 526:143–146[CrossRef][Medline]
  18. Demirgoren S, Majewska MD, Spivak CE, London ED 1991 Receptor binding and electrophysiological effects of dehydroepiandrosterone sulfate, an antagonist of the GABAA receptor. Neuroscience 45:127–135[CrossRef][Medline]
  19. Leranth C, MacLusky NJ, Sakamoto H, Shanabrough M, Naftolin F 1985 Glutamic acid decarboxylase-containing axons synapse on LHRH neurons in the rat medial preoptic area. Neuroendocrinology 40:536–539[Medline]
  20. Jansen HT, Cutter C, Hardy S, Lehman MN, Goodman RL 2003 Seasonal plasticity within the gonadotropin-releasing hormone (GnRH) system of the ewe: changes in identified GnRH inputs and in glial association. Endocrinology 144:3663–3676[Abstract/Free Full Text]
  21. DeFazio RA, Heger S, Ojeda SR, Moenter SM 2002 Activation of GABAA receptors excites gonadotropin-releasing hormone neurons. Mol Endocrinol 16:2872–2891[Abstract/Free Full Text]
  22. Sim JA, Skynner MJ, Pape JR, Herbison AE 2000 Late postnatal reorganization of GABA(A) receptor signalling in native GnRH neurons. Eur J Neurosci 12:3497–3504[CrossRef][Medline]
  23. Spergel DJ, Ulrich D, Hanley DF, Sprengel R, Seeburg PH 1999 GABA- and glutamate-activated channels in green fluorescent protein-tagged gonadotropin-releasing hormone neurons in transgenic mice. J Neurosci 19:2037–2050[Abstract/Free Full Text]
  24. Bilger M, Heger S, Brann DW, Paredes A, Ojeda SR 2001 A conditional tetracycline-regulated increase in {gamma} aminobutyric acid production near luteinizing hormone-releasing hormone nerve terminals disrupts estrous cyclicity in the rat. Neuroendocrinology 142:2102–2114
  25. Jarry H, Perschl A, Wuttke W 1988 Further evidence that preoptic anterior hypothalamic GABergic neurons are part of the GnRH pulse and surge generator. Acta Endocrinol (Copenh) 118:573–579[Abstract/Free Full Text]
  26. Robinson JE 1995 {gamma} Amino-butyric acid and the control of GnRH secretion in sheep. J Reprod Fertil Suppl 49:221–230[Medline]
  27. Terasawa E, Luchansky LL, Kasuya E, Nyberg CL 1999 An increase in glutamate release follows a decrease in {gamma} aminobutyric acid and the pubertal increase in luteinizing hormone releasing hormone release in the female rhesus monkey. J Neuroendocrinology 11:275–282[CrossRef][Medline]
  28. Donoso AO, Lopez FJ, Negro-Vilar A 1992 Cross-talk between excitatory and inhibitory amino acids in the regulation of luteinizing hormone-releasing hormone secretion. Endocrinology 131:1559–1561[Abstract/Free Full Text]
  29. Kaila K 1994 Ionic basis of GABAA receptor channel function in the nervous system. Rev Prog Neurobiol 42:489–537
  30. Huang RQ, Dillon GH 2002 Functional characterization of GABA(A) receptors in neonatal hypothalamic brain slice. J Neurophysiol 88:1655–1663[Abstract/Free Full Text]
  31. Vicini S, Losi G, Homanics GE 2002 GABA(A) receptor {delta} subunit deletion prevents neurosteroid modulation of inhibitory synaptic currents in cerebellar neurons. Neuropharmacology 43:646–450[CrossRef][Medline]
  32. Wohlfarth KM, Bianchi MT, Macdonald RL 2002 Enhanced neurosteroid potentiation of ternary GABA(A) receptors containing the delta subunit. J Neuroscience 22:1541–1549[Abstract/Free Full Text]
  33. Davies PA, Hanna MC, Hales TG, Kirkness EF 1997 Insensitivity to anesthetic agents conferred by a class of GABA(A) receptor subunit. Nature 385:820–823[CrossRef][Medline]
  34. Leidenheimer NJ, Chapell R 1997 Effects of PKC activaiton and receptor desensitization on neurosteroid modulation of GABA(A) receptors. Mol Brain Res 52:173–181[Medline]
  35. Koksma J, van Kesteren R, Rosahl T, Zwart R, Smit AB, Luddens H, Brussaard AB 2003 Oxytocin regulates neurosteroid modulation of GABAA receptors in supraoptic nucleus around partuition. J Neurosci 23:788–797[Abstract/Free Full Text]
  36. Majewska MD, Harrison NL, Schwartz RD, Barker JL, Paul SM 1986 Steroid hormone metabolites are barbituate-like modulators of the GABA receptor. Science 232:1004–1007[Abstract/Free Full Text]
  37. Twyman RE, Macdonald RL 1992 Neurosteroid regulation of GABAA receptor single-channel kinetic properties of mouse spinal cord neurons in culture. J Physiol 456:215–245[Abstract/Free Full Text]
  38. Liu Q, Chang Y, Schaffner A, Smith S, Barker JL 2001 Allopregnanolone activates GABAA receptor/Cl- channels in a multiphasic manner in embryonic rat hippocampal neurons. J Neurophysiol 88:1147–1158
  39. Schmid G, Sala R, Bonanno G, Raiteri M 1998 Neurosteroids may differentially affect the function of two native GABA(A) receptor subtypes in the rat brain. Naunyn Schmiedebergs Arch Pharmacol 357:401–407[CrossRef][Medline]
  40. Genazinni AR, Palumbo MA, de Micheroux AA, Artini PG, Criscuolo M, Ficarra G, Guo AL, Benelli A, Bertolini A, Pertraglia F 1995 Evidence for a role for the neurosteroid allopregnanolne in the modulation of reproductive function in female rats. Eur J Endocrinol 133:375–380[Abstract/Free Full Text]
  41. Kowalski W, Chatterton RTJ 1992 Effects of subchronic infusion of dehydroepiandrosterone sulfate on serum gonadotropin levels and ovarian function in the cynomolgus monkey. Fertil Steril 57:912–920[Medline]
  42. Suter KJ, Song WJ, Sampson TL, Wuarin JP, Saunders JT, Dudek FE, Moenter SM 2000 Genetic targeting of green fluorescent protein to gonadotropin-releasing hormone neurons: characterization of whole-cell electrophysiological properties and morphology. Endocrinology 141:412–419[Abstract/Free Full Text]
  43. Nunemaker CS, DeFazio RA, Moenter SM 2002 Estradiol-sensitive afferents modulate long-term episodic firing patterns of GnRH neurons. Endocrinology 143:2284–2292[Abstract/Free Full Text]
  44. Sullivan SD, DeFazio RA, Moenter SM Central metabolic regulation of fertility through pre- and post-synaptic signaling to gonadotropin-releasing hormone neurons. J Neurosci, in press
  45. DeFazio RA, Moenter SM 2002 Estradiol feedback alters potassium currents and firing properties of gonadotropin-releasing hormone neurons. Mol Endocrinol 16:2255–2265[Abstract/Free Full Text]
  46. Barry PH 1994 JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements. J Neurosci Methods 51:107–116[CrossRef][Medline]
  47. Pitler T, Alger B 1992 Cholinergic excitation of GABAergic interneurons in the rat hippocampal slice. J Physiol 450:127–142[Abstract/Free Full Text]
  48. DeFazio RA, Hablitz JJ 1998 Zinc and zolpidem modulate mIPSCs in rat neocortical pyramidal neurons. J Neurophysiol 80:1670–1677[Abstract/Free Full Text]
  49. Pape JR, Skynner MJ, Sim JA, Herbison AE 2001 Profiling {gamma}-aminobutyric acid (GABA(A)) receptor subunit mRNA expression in postnatal gonadotropin-releasing hormone (GnRH) neurons of the male mouse with single cell RT-PCR. Neuroendocrinology 74:300–308[CrossRef][Medline]
  50. Mitsushima D, Hei D, Terasawa E 1994 {gamma}-Aminobutyric acid is an inhibitory neurotransmitter restricting the release of luteinizing hormone-releasing hormone before the onset of puberty. Proc Natl Acad Sci USA 91:395–399[Abstract/Free Full Text]
  51. Adler BA, Crowley WR 1986 Evidence for {gamma}-aminobutyric acid modulation of ovarian hormonal effects on luteinizing hormone secretion and hypothalamic catecholamine activity in the female rat. Endocrinology 118:91–97[Abstract/Free Full Text]
  52. Piu G, Mienville JM, Matsumoto K, Takahata H, Watanabe H, Costa E, Guidotti A 2003 On the putative physiological role of allopregnanolone on GABAA receptor function. Neuropharmacology 44:49–55[CrossRef][Medline]
  53. Shen W, Mennerick S, Zorumski EC, Covey DF, Zorumski CF 1999 Pregnanolone sulfate and dehydroepiandrosterone sulfate inhibit GABA-gated chloride currents in Xenopus oocytes expressing picrotoxin-insensitive GABAA receptors. Neuropharmacology 38:267–271[CrossRef][Medline]
  54. El-Etr M, Akwa Y, Fiddes RJ, Robel P, Baulieu EE 1995 A progesterone metabolite stimulates the release of gonadotropin-releasing hormone from GT-1 hypothalamic neurons via the {gamma}-aminobutyric acid type A receptor. Proc Natl Acad Sci USA 92:3769–3773[Abstract/Free Full Text]
  55. Mukai H, Uchino S, Kawato S 2000 Effects of neurosteroids on Ca2+ signaling mediated by N-methyl-D-aspartate receptor expressed in Chinese hamster ovary cells. Neurosci Lett 282:93–96[CrossRef][Medline]
  56. Ffrench-Mullen JMH, Spence KT 1991 Neurosteroids block Ca2+ channel current in freshly isolated hippocampal CA1 neurons. Eur J Pharmacol 202:269–272[CrossRef][Medline]
  57. Lagrange AH, Ronnekleiv OK, Kelly MJ 1995 Estradiol-17ß and mu-opioid peptides rapidly hyperpolarize GnRH neurons: a cellular mechanism of negative feedback? Endocrinology 136:2341–2344[Abstract]
  58. Kusano K, Fueshko S, Gainer H, Wray S 1995 Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures. Proc Natl Acad Sci USA 92:3918–3922[Abstract/Free Full Text]
  59. Spergel DJ, Catt KJ, Rojas E 1996 Immortalized GnRH neurons express large-conductance calcium-activated potassium channels. Neuroendocrinology 63:101–111[Medline]
  60. Jorge JC, McIntyre KL, Henderson LP 2002 The function and the expression of forebrain GABA(A) receptors change with hormonal state in the adult mouse. J Neurobiol 50:137–149[CrossRef][Medline]
  61. Herbison AE, Fenelon VS 1995 Estrogen regulation of GABAA receptor subunit mRNA expression in preoptic area and bed nucleus of the stria terminalis of female rat brain. J Neurosci 15:2328–2337[Abstract]
  62. Brussaard AB, Devay P, Leyting-Vermeulen JL, Kits KS 1999 Changes in properties and neurosteroid regulation of GABAergic synapses in the supraoptic nucleus during the mammalian female reproductive cycle. J Physiol 516:513–524[Abstract/Free Full Text]
  63. Corpéchot C, Collins BE, Carey MP, Tsouros A, Robel P, Fry JP 1997 Brain neurosteroids during the mouse oestrous cycle. Brain Res 766:276–280[CrossRef][Medline]
  64. Palumbo MA, Salvestroni C, Gallo R, Guo AL, Genazzani AD, Artini PG, Petraglia F, Genazzani AR 1995 Allopregnanolone concentration in hippocampus of prepubertal rats and female rats throughout estrous cycle. J Endocrinol Invest 18:853–856[Medline]
  65. Goodman RL, Bittman EL, Foster DL, Karsch FJ 1981 The endocrine basis of the synergistic suppression of luteinizing hormone by estradiol and progesterone. Endocrinology 109:1414–1417[Abstract/Free Full Text]
  66. Sullivan SD, Moenter SM 2003 GABAergic mediation of progesterone and androgen feedback to gonadotropin-releasing hormone (GnRH) neurons. Biol Reprod 68(Suppl 1):316 (Abstract)
  67. Pastor CL G-KM, Aloi JA, Evans WS, Marshall JC 1998 Polycystic ovary syndrome: evidence for reduced sensitivity of the gonadotropin-releasing hormone pulse generator to inhibition by estradiol and progesterone. J Clin Endocrinol Metab 83:582–590[Abstract/Free Full Text]
  68. Chang RG, Katz SE 1999 Diagnosis of polycystic ovary syndrome. Endocrinol Metab Clin N Am 28:397–408[CrossRef][Medline]
  69. Biggio G, Purdy RH 2001 Neurosteroids and brain function. San Diego: Academic Press
  70. Eagleson CA, Gingrich MB, Pastor CL, Arora TK, Burt CM, Evans WS, Marshall JC 2000 PCOS: evidence that flutamide restores sensitivity of the GnRH pulse generator to inhibition by estradiol and progesterone. J Clin Endocrinol Metab 85:4047–4052[Abstract/Free Full Text]
  71. Dumesic DA, Abbott DH, Eisner JR, Goy RW 1997 Prenatal exposure of female rhesus monkeys to testosterone propionate increases serum luteinzining hormone levels in adulthood. Fertil Steril 67:155–163[CrossRef][Medline]
  72. Robinson JE, Forsdike RA, Taylor JA 1999 In utero exposure of female lambs to testosterone reduces sensitivity of the gonadotropin-releasing hormone neuronal network to inhibition by progesterone. Endocrinology 140:5797–5805[Abstract/Free Full Text]
  73. Loucks TL, Rohan LC, Kalro BN, Berga SL Increased GABA-amino-butyric acid (GABA) levels in lean women with polycystic ovary syndrome (PCOS). Program of the 84th Annual Meeting of The Endocrine Society, San Francisco, CA, 2002, pp 108–109 (Abstract OR33-1)



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