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¹ Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire, 03755, USA
² Neuroscience Research Centre, Merck Sharp & Dohme Research Laboratories, Harlow, Essex CM20 2QR, UK

	Abstract

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GABAergic transmission regulates the activity of gonadotrophin-releasing hormone (GnRH) neurons in the preoptic area/hypothalamus that control the onset of puberty and the expression of reproductive behaviours. One of the hallmarks of illicit use of anabolic androgenic steroids (AAS) is disruption of behaviours under neuroendocrine control. GnRH neurons are among a limited population of cells that express high levels of the -subunit of the GABA_A receptor. To better understand the actions of AAS on neuroendocrine mechanisms, we have characterized modulation of GABA_A receptor-mediated currents in mouse native GnRH neurons and in heterologous cells expressing recombinant ₂ß₃-receptors. GnRH neurons exhibited robust currents in response to millimolar concentrations of GABA and a picrotoxin (PTX)-sensitive, bicuculline-insensitive current that probably arises from spontaneous openings of GABA_A receptors. The AAS 17-methyltestosterone (17-MeT) inhibited spontaneous and GABA-evoked currents in GnRH neurons. For recombinant ₂ß₃-receptors, 17-MeT inhibited phasic and tonic GABA-elicited responses, accelerated desensitization and slowed paired pulse response recovery. Single channel analysis indicated that GABA-evoked events could be described by three open dwell components and that 17-MeT enhanced residence in the intermediate dwell state. This AAS also inhibited a PTX-sensitive, spontaneous current (open probability, 0.15–0.2) in a concentration-dependent fashion (IC₅₀ 9 µM). Kinetic modelling indicated that the inhibition induced by 17-MeT occurs by an allosteric block in which the AAS interacts preferentially with a closed state and promotes accumulation in that state. Finally, studies with a G302S mutant -subunit suggest that this residue within the transmembrane domain TM2 plays a role in mediating AAS binding and modulation. In sum, our results indicate that inclusion of the -subunit significantly alters the profile of AAS modulation and that this allosteric inhibition of native GnRH neurons should be considered with regard to AAS disruption of neuroendocrine control.

(Received 31 January 2006; accepted after revision 2 March 2006; first published online 16 March 2006)
Corresponding author L. P. Henderson: Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire, 03755, USA. Email: leslie.henderson{at}dartmouth.edu

	Introduction

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Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone that are valuable therapeutic agents, but whose clinical use has been overshadowed by illicit self-administration of supratherapeutic doses. In contrast to physiological levels of endogenous steroids, which are in the nanomolar range (for review, see Shahidi, 2001), serum levels of AAS have been estimated to reach micromolar concentrations in human subjects who abuse them (Masonis & McCarthy, 1995; Wu, 1997; Daly et al. 2001). One of the hallmarks of AAS use is disruption of puberty and of the expression of normal reproductive behaviours (for review, see Clark & Henderson, 2003). These behaviours are regulated by gonadotrophin-releasing hormone (GnRH) neurons located in the preoptic area/hypothalamus which are under a tonic inhibitory tone exerted by GABA_A receptors (for review, see Herbison et al. 1991; Moenter et al. 2003). A critical and unaddressed question is whether AAS alter GABAergic control of GnRH neurons and thus neuroendocrine regulation.

The native GABA_A receptor is a pentameric ionotropic transmembrane protein for which 16 different receptor subunit genes (_1–6, ß_1–3, _1–3, , , and ) and numerous alternatively spliced mRNAs have been identified in mammals (for review, see Whiting et al. 1999). The -subunit shows a highly restricted pattern of expression (Davies et al. 1997; Whiting et al. 1997; Moragues et al. 2000; Sinkkonen et al. 2000) and in rodents is enriched in the ventromedial nucleus of the hypothalamus, the medial preoptic area (mPOA), the septum and the amygdala (Moragues et al. 2000, 2002, 2003; Sinkkonen et al. 2000; McIntyre et al. 2002); all regions intimately involved in the generation of reproductive behaviours (for review, see Clark & Henderson, 2003). Of particular relevance, virtually all GnRH neurons in the preoptic region express marked levels of this subunit (Moragues et al. 2003).

Recombinant receptors composed of ₁ß₃- or ₂ß₁-subunits are both spontaneously active and gated by GABA (Neelands et al. 1999; Davies et al. 2001; Maksay et al. 2003; Wagner et al. 2005). All reports to date indicate that -containing receptors are not sensitive to nanomolar concentrations of high-affinity benzodiazepine (BZ) binding site agonists (Davies et al. 1997, 2001; Whiting et al. 1997; Thompson et al. 1998; Neelands et al. 1999; Maksay et al. 2003), but micromolar concentrations of BZs have been reported to inhibit these -containing receptors (Maksay et al. 2003; cf. Neelands et al. 1999), potentially at a separate low-affinity site. The ability of anaesthetics and anaesthetic neurosteroids to potentiate -containing channels has been somewhat controversial (Davies et al. 1997, 2001; Whiting et al. 1997; Thompson et al. 1998), but the percentage potentiation elicited by these modulators has been shown to be inversely proportional to the level of -subunit expression in recombinant systems (Thompson et al. 2002). Similarly, expression of the -subunit in native neurons has been correlated with diminished sensitivity to BZs (Kasparov et al. 2001), neurosteroids (Jorge et al. 2002) and anaesthetics (Irnaten et al. 2002; Sergeeva et al. 2005).

Anabolic androgenic steroids are both structurally and functionally distinct from the neurosteroids (for review, see Lambert et al. 1995, 2003; Clark et al. 2004). The AAS 17-MeT acts as a positive modulator of currents elicited by application of millimolar GABA to recombinant ₂ß₃₂-receptors, but is without effect on such currents elicited at ₁ß₃₂- or ₂ß₃-receptors. Conversely, 17-MeT potentiates tonic currents elicited by micromolar concentrations of GABA at ₁ß₃₂-receptors, but is without effect on tonic currents produced by stationary levels of micromolar GABA at ₂ß₃₂-receptors. Thus the profile of allosteric modulation elicited by this AAS is dependent upon both subunit composition and the concentration/duration of GABA exposure (Yang et al. 2002, 2005; Clark et al. 2004). GnRH neurons are highly heterogeneous in GABA_A receptor subunit expression (Sim et al. 2000; Todman et al. 2005). While the complement of surface receptors in GnRH neurons is not known, the preferential expression of ₂- and ß₃-subunits in the preoptic area/hypothalamus (Wisden et al. 1992; Herbison & Fénelon, 1995; Pirker et al. 2000; McIntyre et al. 2002; Penatti et al. 2005), coupled with the abundant expression of the -subunit in GnRH neurons (Moragues et al. 2003), suggests that ₂ß₃-receptors are likely to constitute an important receptor class in these cells.

Assessments of GABAergic control of GnRH neurons have been facilitated by the generation of lines of transgenic mice in which green fluorescent protein (GFP) is expressed from the GnRH promoter (Spergel et al. 1999; Suter et al. 2000; Han et al. 2004). Here, we have taken advantage of one of these transgenic lines (Suter et al. 2000) to assess how the commonly abused AAS 17-MeT modulates GABA_A receptor-mediated phasic and spontaneous currents in GnRH neurons. To further define the mechanism of AAS action at -containing receptors, we have assessed modulation by 17-MeT of both GABA-gated and spontaneous currents through recombinant ₂ß₃-receptors to characterize how these abused steroids may influence GABAergic activity in GnRH neurons.

	Methods

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GFP-GnRH transgenic mice and primary neuron preparation

GFP-GnRH mice were generously provided by Dr Suzanne Moenter (Department of Medicine and Cell Biology, University of Virginia, Charlottesville, VA, USA). In these mice, a portion of the mouse GnRH promoter was used to drive expression of enhanced GFP (Suter et al. 2000). Briefly, a portion of the mouse GnRH promoter (–3446 to +23) was used to drive expression of a transgene consisting of the B intron of rabbit ß-globin as a splice donor/acceptor, the coding sequence for enhanced GFP and the polyadenylation signal from human growth hormone. The line was generated in a CBB6 strain, and 99.5% of neurons expressing GFP were found to be GnRH positive (Suter et al. 2000). For experiments performed here, mice were killed with a rising concentration of CO₂, decapitated, and their brains removed directly into an artificial cerebral spinal fluid (ACSF; mM): 124 NaCl, 2.4 CaCl₂, 10 D-glucose, 5 KCl, 1.3 KH₂PO₄, 1.3 MgSO₄ and 24 NaHCO₃, superfused with 95% O₂–5% CO₂. The brain was transected coronally at the level of the optic chiasm, and 400 µm slices were made in this oxygenated ACSF from the rostral portion of the brain using an Electron Microscopy Sciences OTS-4000® vibroslicer (Hatfield, PA, USA). Slices were transferred to a sterile 100 mm Petri dish containing 5 ml of Hibernate-A (BrainBits, Springfield, IL, USA), a Mops-buffered medium designed for use in ambient O₂, which was supplemented with 1 x B27 (Invitrogen Corp., Gaithersburg, MD, USA). Regions containing the mPOA were dissected and incubated in a solution of 2 mg ml^–1 papain (Worthington Biochemical Corp., Lakewood, NJ, USA) in Hibernate-A at 30°C for 30 min. Tissue was then treated with 1.2 U µl^–1 Dnase I type II (Sigma-Aldrich Co., St Louis, MO, USA) and triturated, and the resulting cell suspension was centrifuged for 2 min at 450 x g. The supernatant was decanted, the pellet was resuspended in Neurobasal-A medium (Invitrogen Corp.) supplemented with B27, and the cells were plated onto plastic dishes and maintained at 37°C and 5% CO₂ for at least 1 h prior to recording.

All animal care procedures were approved by Institutional Animal Care and Use Committee at Dartmouth. Procedures were performed to minimize the use of animals and any pain or discomfort to them, and are in agreement with the guidelines and recommendations of the National Institutes of Health and the American Veterinary Medical Association.

Recombinant GABA_A receptor cDNAs and expression in heterologous cells

Transfections and cell maintenance.  Human Embryonic Kidney (HEK) 293 cells (American Type Culture Collection, Manassas, VA, USA) were grown on tissue culture dishes (BD Falcon, Franklin Lakes, NJ, USA) and maintained in Dulbecco's Modified Eagle's Medium (Invitrogen Corp.) supplemented with 10% fetal bovine serum (Invitrogen Corp.), 2 mM L-glutamine, 50 IU ml^–1 penicillin and 50 µg ml^–1 streptomycin (all from Mediatech Inc., Herndon, VA, USA) at 37°C and in 5% CO₂–95% O₂. Constructs encoding the rat ß₃- and _2L- and the human ₂-subunits were kindly provided by Dr Stefano Vicini (Georgetown University Medical School, Washington, DC, USA). The human -subunit cDNA was provided by Dr Paul Whiting (Merck, Sharp & Dohme, Harlow, UK). HEK293 cells were transiently transfected according to Yang et al. (2002, 2005) at equal ratios of 0.8 µg of each plasmid using the Lipofectamine PLUS® protocol (Invitrogen Corp.) for HEK293 cells. The pGreenLantern plasmid (Invitrogen Corp.) was cotransfected (0.8 µg) to permit selection of transfected cells expressing GFP with fluorescence optics. After transfection, cells were maintained in culture medium containing 20 µM bicuculline methiodide (Sigma-Aldrich Co.), which promoted cell survival, and recordings were made approximately 24–48 h after transfection.

Mutagenesis of GABA_A receptor cDNAs.  The mutant G302S GABA_A receptor construct was engineered from the cDNA encoding the human -subunit subcloned into the pCDM8 expression vector (Invitrogen Corp.) using the QuickChange System (Stratgene, La Jolla, CA, USA) and according to the manufacturer's instructions. Numbering of the amino acid 302 refers to position relative to the start of the mature peptide. Mutant DNA was amplified by transformation of XL1-Blue supercompetent cells, transformed colonies were identified, and plasmid DNA was isolated using the Qiagen MiniPrep system (Valencia, CA, USA). Successful mutations were identified by sequencing in both forward and reverse directions using SP6 and T7 primers, respectively. Primers were purchased from Integrated DNA Technologies (Coralville, IA, USA).

The (G302S) primers were: forward, 5'-CTG ACC ATG ACC ACG TTG AGC ACC TTT TCT CG-3'; and reverse, 5'-CGA GAA AAG GTG CTC AAC GTG GTC ATGGTC AG-3'.

Data acquisition and analysis

Pipette fabrication and general recording conditions.  Patch pipettes used for all recordings were fabricated from borosilicate glass (Sutter Instrument Co., Novato, CA, USA). Typically, electrodes used for whole-cell recordings had open tip resistances between 3 and 5 M. Data were acquired and analysed using an EPC-9 patch clamp amplifier and HEKA Pulse software (Instrutech Corp., Port Washington, NY, USA). Whole-cell and nucleated patch recordings were made at room temperature (22–24°C) with a pipette solution containing (mM): 140 CsCl, 1 MgCl₂, 10 Hepes, 5 EGTA and 4 Mg-ATP (pH 7.2, with CsOH). A bath solution composed of the following (mM): 130 NaCl, 6 KCl, 2 MgCl₂, 1 CaCl₂, 10 Hepes, 10 D-glucose and 10 sucrose (pH 7.4, with NaOH) was used for all recordings.

Ultrafast perfusion.  GABA and other modulators were applied to the patch using the LSS-3100 High Speed Positioning System (Burleigh Instruments Inc., Fishers, NY, USA) and a double-barrelled length of theta-glass (Sutter Instruments Co.; tip diameter 100–120 µm) as previously described (Yang et al. 2002, 2005). Open tip currents showed that the 10–90% of the peak on- and off-response was achieved in less than 1 ms. Fast perfusion of transfected HEK293 cells was made using nucleated outside-out patches. Fast perfusion of primary neurons was made using the whole-cell configuration because these acutely isolated cells did not tightly adhere to the dish surface, which made patch excision difficult. All primary neurons were of small diameter, were bereft of major processes following dissociation and had comparable surface area to nucleated outside-out patches, with an average capacitance of 4.6 ± 0.2 pF for the GnRH neurons and 6.9 ± 0.4 pF for the nucleated outside-out patches from HEK293 cells. For experiments examining modulation of GABA-evoked responses, modulators (17-MeT or zolpidem) were present in both streams from the theta-glass in order to pre-equilibrate the patches. When switching between control, drug and wash conditions, or between different concentrations of modulators, a minimum of 2 min was allowed to elapse to ensure full solution exchange. The ultrafast perfusion set-up was plumbed with Teflon^® tubing (Small Parts, Inc. Miami Lakes, FL, USA).

Dish perfusion.  For experiments examining the effects of AAS on currents induced by tonic exposure to GABA, HEK293 cells were transfected and replated at low density, and recordings were made in the whole-cell configuration 5–8 h after replating. Modulators were applied to the bath by gravity flow from a series of manually controlled reservoirs connected with polyethylene tubing to a low volume perfusion manifold (Automate Scientific, San Francisco, CA, USA). Dish fluid volume was kept low (< 500 µl) to minimize total solution exchange time (20 s).

Single channel recordings.  Single channel activity was acquired based on the methods of Steinbach & Akk (2001). Briefly, recordings were made in the on-cell configuration with a pipette solution containing (mM): 125 KCl, 20 TEA-Cl, 10 MgCl₂, 0.1 CaCl₂, 10 D-glucose and 10 Hepes (pH 7.4, with KOH) at a pipette potential (V_pip) of +80 mV. In separate experiments, resting potentials measured in HEK293 cells immediately after establishing a whole-cell configuration were found to be –20 to –40 mV. Therefore we assume that the total membrane potential for experiments in the cell-attached configuration was –100 to –120 mV. When GABA or allosteric modulators were used, they were dissolved in the pipette solution. Patch pipettes for single channel recording had resistances of 8–10 M. Recordings were filtered at 10 kHz and stored to VHS tapes at 100 kHz using a VR-10B pulse code module (Instrutech Corp.). For subsequent analysis, recordings were digitized at 50 kHz using Acquire software (Bruxton Corp., Seattle, WA, USA). Single channel events were detected after digital filtering to 3 kHz using the half-amplitude detection algorithm in QuB (http://www.qub.buffalo.edu), and event files were exported for analysis using TacFit software (Bruxton Corp.). The system dead and rise times were filter-limited (60 and 111 µs, respectively), and the distributions of dwell times were corrected for spuriously short events according to Colquhoun & Sigworth (1995). Analyses were restricted to portions of the recording with no evidence of multiple concurrent channel openings within 500 ms. Subconductance openings (which may have gone undetected using the 50% threshold) accounted for only a small percentage (< 2%) of events detected by the experimenter. Events collected from several patches under the same conditions were pooled before dwell time analysis. Dwell time distributions were fitted using a maximum-likelihood procedure employing correction for missed events (Colquhoun & Sakmann, 1985). Additional kinetic components were added until there was no significant change in the log-likelihood of the fit (Colquhoun & Sigworth, 1995). Assessment of closed duration dwell components were well fitted by four or five components where the longest closures (₃ through ₅) may reflect closure of one receptor followed by sojourns to an open state of a different receptor, as well as long-lived desensitized states of a single receptor. Because these alternatives cannot be differentiated without knowledge of the numbers of channels in the patch (Colquhoun & Sakmann, 1985), long closures are likely to be underestimated. Bursts of openings that exclude these longest-lived closed states are, however, believed to reflect the activity of a single receptor (Sakmann et al. 1980; Colquhoun & Sakmann, 1985; Gibb & Colquhoun, 1991, 1992; Akk et al. 2001; Steinbach & Akk, 2001; Akk & Steinbach, 2003). Bursts were identified as groups of openings separated by a critical duration (T_crit = 17 ms) defined by the method of equal misclassifications (Colquhoun & Sakmann, 1985) such that short and intermediate gaps (closed times represented by ₁ and ₂) were classified as being within a burst (Colquhoun & Sakmann, 1985; Gibb & Colquhoun, 1991, 1992). The resulting burst distributions were fitted as described for open dwell time distributions.

Spectral analysis.  For spectral analysis, recordings were digitized at 100 kHz through a 6 kHz Butterworth filter, and 250 sweeps of 4096 points each were captured. The fast Fourier transform (FFT) of each sweep was calculated after applying a tapered cosine window, and the resulting 250 individual power spectra were averaged. Both control and AAS spectra were readily fitted with two Lorentzian (1/f²) components (Colquhoun & Hawkes, 1977; Korn & Horn, 1988). Time constants (_i) were calculated from corner frequencies (f_c) by: _i = 1/(2 f_ci).

Kinetic modelling

Simulation of channel activity was used to determine kinetic transitions altered by AAS. Models were solved using Q-matrix techniques applied to non-stationary conditions (Colquhoun & Hawkes, 1977, 1995) using programs written (Brian L. Jones) in MatLab 6.1 (The Math Works; Natick, MA, USA; Yang et al. 2002, 2005). For spontaneous currents, the effects of 17-MeT were simulated using a simple three-state kinetic scheme (in the absence of AAS) parameterized directly with microscopic rates estimated from macroscopic current data (see Fig. 7): a slowly equilibrating, non-conducting resting state (R) and rapidly equilibrating closed (C) and open (O) states. The rates connecting R to C (1/_Relax and 1/_Tail; 0.43 and 0.39 s^–1, respectively) were derived from the measurements of relaxation of peak inhibition and decay of the tail current. Closing rate () was estimated at 2564 s^–1 from spectral analysis. Opening rate (ß) was adjusted to 900 s^–1 to provide an equilibrium open probability, P_o, of 0.15. The model provided a qualitatively accurate fit to the data for a wide range of estimates of P_o (0.15–0.5). To model the effects of 17-MeT, a fourth AAS-bound state (B) was added. Blocking and unblocking rates (k_block = 2.1 x 10⁵ M^–1 s^–1; k_unblock = 2.8 s^–1) were obtained from the macroscopic data.

View larger version (24K): [in this window] [in a new window]   Figure 7.  Kinetic assessments of macroscopic currents suggest that 17-MeT promotes inhibition of spontaneous openings by an allosteric block A–C, ultrafast perfusion was used to apply a maximally inhibitory concentration of 17-MeT (100 µM). A, representative response illustrating the relaxation of inhibition during a 10 s application of 17-MeT (relax = 2.7 ± 0.3 s; n = 8) and the rebound current following cessation of 17-MeT application (tail = 2.5 ± 0.2 s; n = 8). B, currents elicited by progressively longer applications of 17-MeT (0.5–32 s) were characterized by asymptotically increasing tail currents. C, time course of this increase in Itail expressed as the percentage of Iholdversus the duration of exposure (rebound = 2.8 ± 0.6 s, n = 6–7 per point). D, representative currents elicited by 1000 ms pulses of 17-MeT (1, 3.16, 10 and 31.6 µM). The onset of inhibition could be described by a single exponential component (block) that was dependent on [17-MeT]. The decline in inhibition was fitted as a double exponential with one component (unblock) that describes release from the blocked state (B; see F) and a second fixed component (tail) that describes relaxation to the resting state (R; see F). E, linear regression indicated that the rate of inhibition (1/block) was dependent on concentration (slope = 2.1(1) x 105M–1 s–1; intercept = 2.8 ± 0.3 s–1; r2 > 0.98). The concentration dependence of unblocking (1/unblock) was minimal, and the slope of the fit was constrained to zero (intercept = 2.7 ± 0.2 s–1, n = 5–7 cells per point). F, macroscopic spontaneous current data could be modelled with a simple kinetic scheme parameterized directly with rates estimated from macroscopic current data that included a non-conducting and slowly equilibrating closed or resting state (R), a rapidly equilibrating closed state (C), an open state (O), and an AAS-bound and non-conducting blocked state (B). The rates connecting R to C (1/relax and 1/tail; 0.43 and 0.39 s–1, respectively) were derived from the measurements of relaxation of peak inhibition and decay of the tail current. Closing rate () was estimated at 2564 s–1 from spectral analysis (see Fig. 8). Opening rate (ß) was adjusted to 1300 s–1 to provide a Po of 1.5–0.2. Blocking and unblocking rates were obtained from data as illustrated in D and E (kblock = 2.1 x 105M–1 s–1; kunblock = 2.8 s–1). The three simulations shown are for AAS durations and concentrations of 10 s at 100 µM (G), 0.5–32 s at 100 µM (H) and 500 ms at 10, 31.6 and 100 µM (I) to simulate data shown in A, B and D, respectively.

Stock solutions of zolpidem (N,N-6-trimethyl-2-(4-methylphenyl)-imidazo[1,2-a]pyridine-3-acetamide), 17-MeT (17-methyl-4-androsten-17ß-ol-3-one) and picrotoxin (PTX; Sigma-Aldrich Co.) were made with cell culture grade dimethyl sulphoxide (DMSO) as the solvent and were diluted to achieve a final bath concentration 0.01% DMSO. Control experiments demonstrated that DMSO up to 0.1% had no effect on current properties (data not shown). Bicuculline methiodide (BMI; Sigma-Aldrich Co.) was prepared as an aqueous solution and kept frozen until time of use.

Statistical analysis

Values are reported as means ± S.E.M. For each cell, current parameters in the presence of a modulator (17-MeT or zolpidem) were calculated as the percentage of the parameters measured under control conditions, and statistical significance was assessed using one-way two-tailed student's t tests. One-way ANOVA was used to establish the significance of differences measured under more than two experimental conditions; subsequent pairwise multiple comparisons were performed with student's t tests using the Bonferroni post hoc correction. For direct comparisons of dose–response and binding rate curves, two-way ANOVA with factors of receptor types and concentrations followed by a Bonferroni post hoc correction were used. This method allowed for model-free comparison of these data. This method of analysis is a useful adjunct to comparison of parameters derived from fitting of the empirically derived Hill equation, since the dose–response relationship described by the Hill equation is unlikely to apply to the gating mechanisms of real ion channels (Colquhoun, 1998). The uncertainties reported with parameters derived from curve fitting procedures are the S.E.M. values recovered from the fitting process. One- or two-sample two-tailed independent student's t tests were performed to determine significance between the responses of different combinations of modulators unless otherwise specified.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Properties of GABA_A receptor-mediated currents in GnRH neurons

GABA-evoked responses in GnRH neurons.  To determine the properties of GABA_A receptor-mediated currents in native GnRH neurons, whole-cell recordings were made from acutely dissociated fluorescent neurons derived from a transgenic mouse expressing GFP under the GnRH promoter (Suter et al. 2000). All animals used in these studies were males between the ages of postnatal days 20 and 30. To mimic the time course and concentration parameters of synaptic release, 1 mM GABA was applied for a brief duration (3 ms) using ultrafast perfusion. This application protocol elicited currents from GnRH neurons that were characterized by an average peak current (I_peak) of –2500 ± 500 pA, a maximum current density (_max) of –580 ± 90 pA pF^–1 and a total charge transfer (Q_tot) of –180 ± 20 pC (n = 15) (Fig. 1). Currents deactivated with a tri-exponential decay well described by time constants ₁ = 13 ± 1 ms (43 ± 3% contribution to I_peak), ₂ = 78 ± 6 ms (48 ± 3%) and ₃ = 357 ± 34 ms (11 ± 1%). Decay could also be summarized by a single weighted time constant, _w = 75 ± 7 ms (n = 15; Fig. 1).

View larger version (51K): [in this window] [in a new window]   Figure 1.  GABAA receptor-mediated currents in GnRH neurons A, GFP-GnRH neurons in a 100 µm slice through the mPOA of a male mouse at postnatal day 20. B, representative current evoked by a 3 ms application of 1 mM GABA to a dissociated GnRH neuron (VH = –60 mV). C, dissociated GFP-GnRH neuron in the absence of GABA displayed tonic currents that were blocked by PTX and 17-MeT (500 ms applications), but were unaffected by bicuculline methiodide (BMI).

Inhibition by AAS of GABA_A receptor-mediated currents from GnRH neurons.  As noted above, other well-studied allosteric modulators of the GABA_A receptor have paradoxical effects on responses mediated by -subunit-containing receptors. To assess AAS modulation of currents that are likely to reflect activity of -subunit-containing GABA_A receptors in GnRH neurons, the effect of a high concentration of 17-MeT (10 µM) on I_hold was compared to that of a maximally effective concentration of PTX (100 µM) in the same cell. Application of 17-MeT diminished I_hold by 42 ± 20 pA (10 ± 4 pA pF^–1). When the effects of PTX and 17-MeT were compared within individual cells, the ratio of antagonism of I_hold produced by 10 µM 17-MeT to that produced by 100 µM PTX was 0.4 ± 0.2 (n = 3). These data indicate that 17-MeT acts as a negative modulator of the spontaneous current in GnRH neurons. While this negative modulation may reflect AAS action at -subunit-containing receptors, the extensive heterogeneity in GABA_A receptor subunits expressed in GnRH neurons, especially in juvenile mice (Sim et al. 2000; Todman et al. 2005), introduces variability in assigning properties of AAS modulation in these primary neurons to any specific class of receptor. To better understand the mechanisms by which inclusion of the -subunit affects AAS modulation, experiments were subsequently performed on recombinant ₂ß₃-receptors expressed in HEK293 cells.

Properties of macroscopic currents mediated by recombinant ₂ß₃-receptors

Concentration–response parameters for currents elicited by GABA from ₂ß₃-receptors.  Application of GABA elicited concentration-dependent responses from recombinant ₂ß₃-receptors with an EC₅₀ of 3.9 ± 0.9 µM and a Hill slope of 0.52 ± 0.05 (n = 4; V_H = –60 mV; Fig. 2 and Table 2). While there are no published data for ₂ß₃-receptors, these values are in general agreement with those reported for ₁ß₃-receptors (EC₅₀ of 0.8 µM; Hill slope of 0.8; Neelands et al. 1999) and for ₂ß₁-receptors (EC₅₀ of 11.2 µM; Hill slope of 1.1; Davies et al. 1997). The apparent affinity of GABA for ₂ß₃-receptors was higher than that observed for ₂ß_32L-receptors (EC₅₀ of 17 ± 1 µM; Yang et al. 2005). The lower EC₅₀ of ₂ß₃- versus ₂ß_32L-receptors is in keeping with a role for the -subunit-containing receptors in mediating extrasynaptic and tonic conductances that would most probably be activated in response to low concentrations of GABA.

View larger version (13K): [in this window] [in a new window]   Figure 2.  Phasic and tonic responses elicited by GABA from recombinant 2ß3-receptors A, representative currents elicited by perfusion of a 62.5–1000 ms application of 0.1 µM to 10 mM GABA. B, concentration-response relationship for currents elicited by 125–1000 ms pulses of GABA from 0.1 µM to 10 mM. For each cell, the Ipeak of each current was normalized to the maximal response for that cell. Values represent 4 cells per point.

View this table: [in this window] [in a new window]   Table 2. Properties of current responses and modulation by 17-MeT for wild-type and G302S receptors

View larger version (22K): [in this window] [in a new window]   Figure 3.  Modulation by AAS of GABA-dependent currents through 2ß3-receptors A, representative currents showing the response elicited by a phasic (3 ms) application of 1 mM GABA and the inhibition of Ipeak induced by application of 1 µM 17-MeT. The inhibitory effect of the AAS was reversible (Ipeak during wash > Ipeak during AAS) and Ipeak returned to control levels when scaled for the run-down of the response that occurs in response to prolonged (10–30 min) application of 1 mM GABA alone (not shown). B, representative responses to stationary application of 1 µM GABA and 1 µM 17-MeT, demonstrating reversible inhibition of Itonic by this AAS. C, graphic representation of the effects of 1 µM 17-MeT on parameters of currents elicited by brief (3 ms) pulses of 1 mM GABA (phasic: Ipeak, Atot, w, 1–3,%1–3) and steady-state application of 1 µM GABA (Itonic). *P < 0.05, significantly different from control values.

Modulation by AAS of phasic responses mediated by ₂ß₃-receptors. 

Modulation by AAS of tonic responses mediated by ₂ß₃-receptors.  Other positive allosteric modulators of GABA_A receptors have been shown to enhance GABA-evoked tonic conductances mediated by -subunit-containing receptors selectively (Stell et al. 2003; Wei et al. 2004). For experiments performed here, tonic current was defined as the GABA-activated current elicited by sustained (bath) application of GABA that does not include any contribution from spontaneously opening ₂ß₃-subunit-containing receptors. Bath application of a low concentration of GABA (1 µM) resulted in tonic currents (I_tonic) from recombinant ₂ß₃-subunit-containing receptors of –670 ± 120 pA (n = 8; Fig. 3B). One micromolar 17-MeT significantly inhibited these tonic currents (to 82 ± 3% of control values, P = 0.003, n = 8; Fig. 3B and C). In contrast, 1 µM 17-MeT was without effect on tonic currents elicited by 1 µM GABA from ₂ß_32L-receptors (103 ± 9% of control values, P = 0.79, n = 7), consistent with previous results (Yang et al. 2005). This concentration of 17-MeT was also without effect on tonic currents elicited by 10 µM GABA, which represents a concentration of agonist that is predicted to produce a fractional response at ₂ß_32L-receptors equivalent to 1 µM GABA at ₂ß₃-receptors (data not shown). The high-affinity BZ site agonist zolpidem (1 µM) also induced a small but significant inhibition of I_tonic from ₂ß₃-subunit-containing receptors (to 90 ± 3% of control values, P = 0.02, n = 6). This is in contrast to the potentiation of I_tonic from ₂ß_32L-receptors by 1 µM zolpidem (to 453 ± 46% of control values, P < 0.001).

Modulation by AAS of desensitization, paired pulse recovery and high-frequency responses mediated by ₂ß₃-receptors.  To determine whether AAS alter desensitization of ₂ß₃-subunit-containing receptors, the effect of 17-MeT on currents produced by 500 ms applications of 1 mM GABA was studied (Fig. 4). GABA-evoked currents activated quickly with a 10–90% rise time of 2.1 ± 0.2 ms (n = 10) and were described by an average maximal current (I_GABA) of –1230 ± 240 pA, a _max of 210 ± 31 pA pF^–1 and Q_tot of – 39 ± 32 µC (n = 10). Current desensitization was biphasic in seven out of 10 cells exposed to GABA, with a fast component, _des-f = 29 ± 11 ms (23 ± 6%) and a slow component, _des-s = 521 ± 85 ms (76 ± 6%). For the three cells that showed monophasic desensitization, decay was well described by _des-s. Overall desensitization was described by a single weighted time constant of deactivation (_des) of 410 ± 80 ms (n = 10; Fig. 4A–C). The ratio of steady-state to peak current under these conditions was 34 ± 6% (n = 10).

View larger version (22K): [in this window] [in a new window]   Figure 4.  Anabolic androgenic steroid alters the kinetics of GABA-induced desensitization and slows paired pulse recovery A, currents evoked by 500 ms application of 1 mM GABA show significant desensitization. Exposure to 1 µM 17-MeT reduces Ipeak and accelerates desensitization without altering deactivation. B, currents from A normalized to peak illustrate acceleration of desensitization. Inset: deactivation of representative currents elicited by 500 ms pulses of GABA in the presence and absence of 17-MeT scaled to the current level immediately before the end of application show no differences in kinetics. C, bar graph summarizing AAS-induced changes in deactivation and desensitization parameters. Desensitization during the pulse could be described by a fast (des-f) and a slow component (des-f) or a single weighted time constant, des. Current deactivation following the pulse could also be described by a fast (off-f) and a slow (off-s) component or a single weighted function (off). D, AAS effects on recovery to paired responses. Insets show representative currents elicited by paired 3 ms pulses of 1 mM GABA in the presence and absence of 1 µM 17-MeT. Comparison of the recovery time course of the second response in the paired pulse protocol shows that 1 µM 17-MeT slows response recovery. Percentage recovery of the second pulse (% Recovery), as assessed by (Ipeak2 – onset2)/(Ipeak1 – onset1) x 100, was plotted as a function of interpulse interval and described by two exponential components with time constants, r1 and r2. Pulses with delays of 25–12 800 ms were used for analysis, but currents are only displayed to 3200 ms for clarity. E, exposure to 1 µM 17-MeT inhibited the response to repetitive stimulation, significantly decreasing Qtot. *P < 0.05, significantly different from control values.

Paired 3 ms applications of 1 mM GABA with varied interpulse intervals were used to assess the effect of 17-MeT on the time course of recovery between successive applications of GABA. Ten paired applications with delays from 25 to 12 800 ms (doubling at each interval) were collected. The time course of recovery was bi-exponential with time constants of _r1 = 95 ms (85%) and _r2 = 2660 ms (15%; n = 10; Fig. 4D). Reponses recovered to 100% of the initial peak amplitude by 12 800 ms. Analysis of recovery curves by two-way ANOVA permitted a model-free comparison of AAS-induced changes in response recovery and indicated that 17-MeT produced significant slowing of recovery (F_1,9 = 4.43, P = 0.04, n = 7–10 per point) by increasing _r1 to 109 ms (155% of control values) and _r2 to 2450 ms (120% of control values; Fig. 4D).

Recordings made from native GnRH neurons indicate that these cells are subject to a high frequency of GABA_A receptor-mediated synaptic inputs (Sim et al. 2000; Nunemaker et al. 2003). To determine whether AAS exposure might alter overall charge transfer arising from high-frequency phasic responses mediated by -subunit-containing receptors, HEK293 cells expressing ₂ß₃-receptors were exposed to trains (10 pulses, 3 ms in duration and 25 ms delay) of 1 mM GABA. This protocol produced an average net charge transfer during the train (Q_tot) of –4 ± 4 µC. Exposure to 1 µM 17-MeT produced a significant decrease in Q_tot (to 64 ± 10% of control values, P = 0.01, n = 7) (Fig. 4E). This suggests that 1 µM 17-MeT would have a net antagonistic effect on high-frequency phasic transmission mediated by -subunit-containing GABAergic synapses which may alter the episodic firing patterns observed in GnRH neurons (Sim et al. 2000; Nunemaker et al. 2003).

Properties of spontaneous current mediated by ₂ß₃-recombinant receptors.  Anabolic androgenic steroids administered in vivo, even at low doses, have been shown to diminish the release of luteinizing hormone (LH) dramatically (Bronson et al. 1996). The presence of a PTX-sensitive, bicuculline-insensitive current in GnRH neurons suggests that spontaneously active GABA_A receptors may play an important role in regulating the activity of these native neurons and their control of pituitary LH release, and thus may also be an important target with respect to AAS action. To determine how AAS interact with spontaneously active -subunit-containing GABA_A receptors, we first characterized the properties of spontaneous currents mediated by ₂ß₃ recombinant receptors and subsequently examined the effects of 17-MeT on these currents.

Baseline I_hold (–240 ± 40 pA; –31 ± 7 pA pF^–1; V_H = –60 mV) was evident in all cells expressing ₂ß₃-receptors (n = 10; Fig. 5). Picrotoxin (100 µM; 500 ms) reversibly antagonized I_hold (by 65 ± 7%). The portion of I_hold antagonized by PTX reversed at –0.3 ± 1 mV, consistent with the predicted reversal potential for Cl^– under our recording conditions, and showed subtle outward rectification with a ratio of 1.24 ± 0.08 for I_hold(+60mv):I_{hold(–60mV)} (Fig. 5D; n = 10). Taken together, these data indicate that I_hold arises from spontaneous openings of GABA_A receptors expressed in the HEK293 cells.

View larger version (22K): [in this window] [in a new window]   Figure 5.  Recombinant 2ß3-receptors in HEK293 cells open in the absence of GABA A, recombinant 2ß3-receptors were activated by 1 mM GABA, but also displayed a notable level of spontaneous current in the absence of GABA that could be blocked by 100 µM of the non-competitive GABAA antagonist, PTX. Blockade of spontaneous openings appears as an outward deflection in Ihold (VH = –60 mV). B, voltage steps from –60 to +60 mV in control saline (bath) or in saline supplemented with 100 µM PTX (+PTX) show that application of PTX is associated with a decrease in Ihold at all potentials. C, membrane conductance at all potentials is decreased by 100 µM PTX. D, in solutions containing symmetrical chloride concentrations, Ihold reverses at 0 mV (Vr). Collectively, these observations indicate that Ihold arises from unliganded openings of GABAA receptors.

An approximation of P_o can also be made from spectral analysis of channel noise (Colquhoun & Hawkes, 1977). In the power spectra of channel noise from recombinant ₂ß₃-receptors, the contribution of the 0.39 ms component, which we believe corresponds to the apparent mean channel open time, is 21 ± 3% (Fig. 8C). Data from these two approaches suggest that the P_o for unliganded ₂ß₃-receptors is 0.15–0.2.

View larger version (21K): [in this window] [in a new window]   Figure 8.  Anabolic androgenic steroid does not alter channel open dwell time A, representative spontaneously opening single channel currents (downward deflections) recorded in the on-cell configuration (Vpip = +80 mV). Most events are < 1 ms; attenuation of peak amplitude by filtering is evident. B, noise currents recorded in the presence or absence of a maximally inhibitory concentration of 17-MeT (100 µM), which decreased mean current level and variance. C, power spectra for AAS and control conditions. Both control and AAS spectra were well described by two Lorentzian components (f and s). D, bar graph summarizing changes in spectral parameters. Anabolic androgenic steroid did not induce a change in the time constant of either the fast (f) or the slow time constant (s), but did decrease the absolute power of both components (Sf and Ss). *P < 0.05, significantly different from control values.

Modulation by AAS of spontaneous current mediated by recombinant ₂ß₃-receptors. 

View larger version (14K): [in this window] [in a new window]   Figure 6.  Dose–response relationship for AAS inhibition of spontaneous GABAA receptor-mediated current A, representative current trace showing dose-dependent inhibition of spontaneous current by 17-MeT. To control for variability in this spontaneous holding current (Ihold), the peak of inhibition was normalized to that produced by a saturating concentration of PTX (100 µM). B, when fitted with the Hill equation, these data gave half-maximal inhibition (IC50) of 15 ± 7 µM and a Hill slope of 0.61 ± 0.07. Values represent 5–7 cells per point.

To examine the dynamics of inhibition of I_hold by 17-MeT, the rates of inhibition and relaxation of peak inhibition were assessed at 1, 3.16, 10 and 31.6 µM 17-MeT (500 ms). The onset of inhibition was fitted as a single exponential (_block; Fig. 7D). For concentrations less than 1 µM, the signal-to-noise ratio of the recordings was inadequate for curve fitting. For other concentrations, the rate of relaxation of inhibition was assessed by fitting the decline in inhibition (I_tail) with a double exponential function with one component (_unblock) that described release from the blocked state and a second component (_tail) that described relaxation to the resting state (Fig. 7E). This procedure allowed the unblocking rate to be disentangled from the rate of relaxation of I_tail (_tail). The dependence of the rate of blocking (1/_block) on concentration was described by a linear relationship with a slope of 2.1(1) x 10⁵ M^–1 s^–1 and an intercept of 2.8 ± 0.3 s^–1 (r² > 0.98; Fig. 7E). The concentration dependence of unblocking (1/_unblock) was minimal, and the slope of the fit was constrained to zero, producing an intercept of 2.7 ± 0.2 s^–1 (Fig. 7E). A simple bimolecular binding mechanism implies that the slope of the blocking rate curve is the binding rate (k_block) and that the intercept of the blocking rate curve is the unbinding rate (k_unblock; Newland & Cull-Candy, 1992; Fig. 7F).

The linear dependence of 1/_block on concentration is consistent with several mechanisms of inhibition, including physical obstruction of the channel pore (open channel block) and stabilization of a new or existing closed state (allosteric block). However, the inhibition of I_hold produced by 100 µM 17-MeT exhibited a small but significant dependence on voltage, where the block at +60 mV was 88 ± 3% of the block at –60 mV (P = 0.03; n = 4). This observation is not consistent with a mechanism in which 17-MeT acts to physically obstruct the channel pore, since reversal of ion flux might be expected to disturb such an obstruction even for an uncharged molecule. To differentiate between an open channel block and an allosteric block as the mechanism of AAS inhibition more fully, the effects of 17-MeT on single channel properties were assessed.

Properties of single channel currents mediated by recombinant ₂ß₃-subunit-containing receptors

Effects of AAS on open dwell times of spontaneous events.  Open channel block mechanisms reduce channel open dwell time by providing an additional non-conducting state beyond normal closing (Sakmann et al. 1980). In contrast, inhibition of channel activity characterized by a decrease in channel frequency with no change in time constants of burst length is consistent with enhanced occurrence of a desensitized state or an allosterically blocked state (Newland & Cull-Candy, 1992). Ideally, an AAS-dependent change in open dwell time could be detected with single channel analysis; however, the mean open times of spontaneous events mediated by ₂ß₃-subunit-containing receptors were quite brief (Fig. 8A), making a direct single channel assessment of AAS effects on spontaneous events difficult. Consequently, in order to discriminate between an allosteric versus an open channel block mechanism, the effect of a maximally inhibitory concentration of 17-MeT (100 µM) on spontaneous current noise was examined by spectral analysis.

Noise spectra, both under control conditions and in the presence of 17-MeT, were well described by two Lorentzian components with time constants of _I = 0.39 ± 0.08 ms (21 ± 3%) and _s = 2.8 ± 0.3 ms (79 ± 3%; Fig. 8C), suggesting that the 0.39 ms component of the noise spectrum reflects the apparent mean channel open time for spontaneously gated receptors. Values of _f are in agreement with the time constant reflecting the brief component of the open dwell distribution (0.388 ± 0.057 ms; 57%) for ₁ß₃-subunit-containing receptors gated by a low (1 µM) concentration of GABA (Neelands et al. 1999) and the briefest component of the open and burst duration distributions (₁) we report here for currents elicited by 1 mM GABA from ₂ß₃-subunit-containing receptors (Table 1).