Anabolic Steroids Induce Region- and Subunit-Specific Rapid Modulation of GABAA Receptor-Mediated Currents in the Rat Forebrain

Juan Carlos Jorge-Rivera,1 Kerry L. McIntyre,2 and Leslie P. Henderson1,2

Departments of  1Physiology and  2Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Jorge-Rivera, Juan Carlos, Kerry L. McIntyre, and Leslie P. Henderson. Anabolic Steroids Induce Region- and Subunit-Specific Rapid Modulation of GABAA Receptor-Mediated Currents in the Rat Forebrain. J. Neurophysiol. 83: 3299-3309, 2000. Anabolic-androgenic steroids (AAS) have become significant drugs of abuse in recent years with the highest increase reported in adolescent girls. In spite of the increased use of AAS, the CNS effects of these steroids are poorly understood. We report that in prepubertal female rats, three commonly abused AAS, 17alpha -methyltestosterone, stanozolol, and nandrolone, induced rapid and reversible modulation of GABAergic currents in neurons of two brain regions known to be critical for the expression of reproductive behaviors: the ventromedial nucleus of the hypothalamus (VMN) and the medial preoptic area (mPOA). All three AAS significantly enhanced peak synaptic current amplitudes and prolonged synaptic current decays in neurons of the VMN. Conversely all three AAS significantly diminished peak current amplitudes of synaptic currents from neurons of the mPOA. The endogenous neuroactive steroids, 3alpha -hydroxy-5alpha -pregnan-20-one and 5alpha -androstane-3alpha ,17beta -diol, potentiated currents in the VMN as did the AAS. In contrast to the negative modulation induced by AAS in the mPOA, the endogenous steroids potentiated responses in this region. To determine the concentration response relationships, modulation by the AAS, 17alpha -methyltestosterone (17alpha -meT), was assessed for currents evoked by ultrafast perfusion of brief pulses of GABA to acutely isolated neurons. Half-maximal effects on currents elicited by 1 mM GABA were elicited by submicromolar concentrations of AAS for neurons from both brain regions. In addition, the efficacy of 10-5 to 10-2 M GABA was significantly increased by 1 µM 17alpha -meT. Previous studies have demonstrated a striking dichotomy in receptor composition between the VMN and the mPOA with regard to gamma  subunit expression. To determine if the preferential expression of gamma 2 subunit-containing receptors in the VMN and of gamma 1 subunit-containing receptors in the mPOA could account for the region-specific effects of AAS in the two regions, responses elicited by ultrafast perfusion of GABA to human embryonic kidney 293 cells transfected with alpha 2, beta 3, and gamma 2 or alpha 2, beta 3, and gamma 1 subunit cDNAs were analyzed. As with native VMN neurons, positive modulation of GABA responses was elicited for alpha 2beta 3gamma 2 recombinant receptors, while negative modulation was induced at alpha 2beta 3gamma 1 receptors as in the mPOA. Our data demonstrate that AAS in doses believed to occur in steroid abusers can induce significant modulation of GABAergic transmission in brain regions essential for neuroendocrine function. In addition, the effects of these steroids can vary significantly between brain regions in a manner that appears to depend on the subunit composition of GABAA receptors expressed.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Anabolic-androgenic steroids (AAS), synthetic derivatives of testosterone originally designed to provide enhanced anabolic potency with negligible androgenic effects (for review, Kochakian 1993), have become significant drugs of abuse not only among elite athletes, but among a growing number of adolescents (Johnson 1990; Yesalis et al. 1997) especially young girls (Bahkre et al. 1998). It has been noted that long term risks from AAS abuse are greater in women than in men (Franke and Berendonk 1997; Hickson and Kuowski 1986; Honor 1997; Strauss and Yesalis 1993) and that AAS use in both women and female rodents is associated with irregular cyclicity (Blasberg et al. 1997; Bronson 1996; Bronson et al. 1996; Clark et al. 1998a; Franke and Berendonk 1997), accelerated reproductive senescence (Bronson 1996), and changes in both aggressive and sexual behaviors (Bronson 1996; Bronson et al. 1996). While adverse effects of long-term AAS treatment on estrous cyclicity can be attributed to signaling through androgen receptors (Blasberg et al. 1998), recent studies demonstrating that AAS alter Cl- flux in synaptosomes, as well as binding of t-butylbicyclophosphorothionate (TBPS) and benzodiazepines to the gamma -aminobutyric acid type A (GABAA) receptor (Masonis and McCarthy 1995; 1996), suggest that these compounds may have acute effects in the CNS that are mediated by nongenomic actions at the GABAA receptor.

Here we show for the first time that three commonly abused AAS, 17alpha -methyltestosterone (17alpha -meT), stanozolol, and nandrolone, induced rapid modulation of GABAA receptor-mediated synaptic currents in the ventromedial nucleus of the hypothalamus (VMN) and the medial preoptic area (mPOA), two forebrain regions known to play critical, but contrasting, roles in regulating female reproductive behaviors (for review, McCarthy 1995). AAS elicited opposing effects in these two regions, enhancing currents in neurons from the VMN while diminishing them in neurons from the mPOA. Ultrafast application of GABA plus AAS to acutely isolated neurons demonstrated that AAS altered GABA efficacy in a dose-dependent fashion. Moreover, assessment of currents elicited from recombinant receptors in heterologous cells suggested that the opposing pattern of AAS modulation in the two brain regions may arise, at least in part, from the preferential expression of gamma 2 subunit-containing receptors in the VMN and gamma 1 subunit-containing receptors in the mPOA. Finally, we show that the endogenous neuroactive steroids, 3alpha -hydroxy-5alpha -pregnan20-one (allopregnanolone; 3alpha ,5alpha -THP) and 5alpha -androstane-3alpha ,17beta -diol (3alpha -DIOL) also modulated synaptic currents in both regions; however, AAS and the endogenous neuroactive steroids had opposite effects in the mPOA, suggesting different mechanisms of actions for these two classes of steroids at the GABAA receptor.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Animal care and preparation of neuronal tissue

Prepubertal Sprague Dawley female rats [postnatal day PN (3-14)] were employed in this study (n = 245). Animal care procedures were approved by the Institutional Animal Care and Use Committee at Dartmouth and adhere to both the National Institutes of Health and the American Veterinary Medical Association guidelines. For slice recordings, animals were rapidly decapitated, the brains were quickly dissected and placed in ice-cold saline. A thick coronal section was mounted with cyanoacrylic ester (Krazy glue) on the chuck of a Campden Vibroslice microtome (Stoelting, Wood Dale, IL), and 300-µM slices at the level of the VMN or the mPOA were prepared. For isolation of acutely dissociated neurons, the VMN or the mPOA was quickly microdissected from animals of comparable ages. Tissue was minced into small pieces and transferred through several washes and incubated in 0.25% trypsin (Worthington Biochemical, Freehold, NJ) in Opti-MEMI (Gibco Laboratories; Grand Island, NY) at 37°C for 10 min followed by 8-min incubation in 0.2 mg/ml DNase (Sigma Chemical, St. Louis, MO) in the same trypsin-containing medium. Enzyme activity was inhibited by incubation at room temperature in Opti-MEMI containing 5% charcoal-stripped fetal bovine serum (FBS; Gibco) for 8 min. Medium was removed, the tissue triturated, and the cells were plated onto 35-mm tissue-culture dishes coated with Cell-tak (Collaborative Biomedical Products; Bedford, MA). Recordings were made 30-60 min after plating.

Transfection of HEK293 cells

Human alpha 2, rat beta 3, rat gamma 2, and human gamma 1 GABAA receptor subunit cDNAs individually subcloned into the pCDM8 or M13 expression vectors were provided by Dr. Stefano Vicini (Georgetown University Medical Center, Washington, DC). Human embryonic kidney (HEK) 293 cells, provided by Dr. Lee Witters (Dartmouth Medical School, Hanover, NH), were transfected using Lipofectamine (Gibco) with plasmids expressing cDNAs encoding the alpha 2, beta 3, and gamma 2 subunits or with ones expressing cDNAs encoding alpha 2, beta 3, and gamma 1 subunits (1 µg of each construct). Cotransfection of the plasmid, pGreenLantern (Gibco), allowed for selection of transfected cells expressing the green fluorescent protein (GFP) under fluorescent optics.

Acquisition and analysis of spontaneous inhibitory postsynaptic currents (sIPSCs)

Recordings were made as described previously (Nett et al. 1999) using an Olympus BX50 microscope equipped with a Dage VE1000 CCD camera system (Optical Analysis, Nashua, NH) from slices superfused with 95%O2-5%CO2-saturated artificial CSF (ACSF) containing 125 mM NaCl, 4 mM KCl, 26 mM NaHCO3, 2 mM CaCl2, 1 mM MgCl2, and 10 mM glucose supplemented with 10 µM CNQX and 20 µM CPP to block glutamatergic transmission (Smith et al. 1996). Pipette saline consisted of 153 mM CsCl, 1 mM MgCl2, 5 mM EGTA, and 10 mM HEPES to which 2 mM MgATP was added each day. All chemicals were purchased from Sigma with the exception of diazepam (RBI; Natick, MA) and nandrolone (Steraloids; Wilton, NH). Recordings were made at 20-22°C, at a holding potential (VH) of -80 mV. Modulators were dissolved in DMSO (0.01% final concentration) and applied to the bath via gravity flow. Three to 5 min of predrug data were acquired, the bath was then changed to steroid-containing ACSF (1 µM steroid), and 3-5 min later data were again acquired for 3-5 min. The bath was then switched back to ACSF alone, and 3-5 min were allowed to pass before postdrug data were collected. Recordings were made using series resistance compensation of 50-75%. Data were acquired with a List EPC-7 amplifier (ALA Scientific Instruments; Westbury, NY) and a PowerMac 8600 and analyzed using HEKA software (PulseFit; Instrutech; Great Neck, NY) and the Mini Analysis Program (Jaejin Software; Leonia, NJ.). More than 50 sIPSCs with times to peak <2 ms were acquired, averaged, and analyzed for each drug condition for each neuron, and current averages were fitted under conditions in which the number of kinetic components, their magnitudes, and absolute values were not restrained, and the fits were optimized to give root mean square < 5%. No significant correlations were found between time to peak versus the value of tau 1, time to peak versus the value of tau 2, the percentage of the peak current attributed to tau 1 versus the time to peak, or the value of tau 1 versus the percentage of the peak current attributed to tau 1 (linear regression analysis gave multiple R values and slopes close to 0 for all comparisons) for sIPSCs recorded from neurons of these two regions.

Recording and analysis of responses from acutely isolated neurons

Responses were elicited by ultrafast perfusion of GABA or GABA plus steroids for both acutely isolated forebrain neurons and HEK293 cells in the whole cell configuration (VH = -80 mV) using a LSS-3100 high-speed positioning system (Burleigh Instruments, Fishers, NY) and solutions described in the preceding text. Assessment of open tip currents (Lester and Jahr 1992) indicated that 10-90% of the peak on and off responses that reflect time of solution exchange were achieved in <500 µs with this system and that applications were stable with repetitive exposures. Since steroids are known to be difficult to wash both from cell membranes and from tissue culture plastic, data were first acquired from separate populations of cells exposed either to 1 mM GABA alone or exposed simultaneously to 1 mM GABA plus 1 µM 17alpha -meT. Subsequent experiments in which the same, individual cells were first exposed to GABA, then to GABA plus 17alpha -meT, and finally to GABA alone again (wash) using solution switching techniques (Zhu and Vicini 1997) gave comparable results to those obtained from population studies, and the data have been pooled. No differences were noted between neurons exposed to modulator prior to GABA (preequilibrated) and those exposed to GABA and modulator simultaneously. For the experiments presented here, neurons were not preequilibrated with AAS.

Data acquisition and analysis of responses elicited by ultrafast perfusion to either isolated neurons or to HEK293 cells was made as described in the preceding text for synaptic currents with the following modifications. As previously described (Smith et al. 1996), initial assessment of current fits by eye indicated that the majority of responses were not well fitted by two exponential components with the largest error noted in the fastest component of current decay as described by tau 1. Therefore a least-squares fit using the simplex algorithm was generated for three exponential components of current decay with no restraints imposed on any individual component for each elicited response. Plots of all time constants indicated the presence of three distributions of time constants and the ranges of values for each distribution. Individual responses that, when initially fitted with three components, had more than one time constant that fell within a single distribution were subsequently refitted with two components. For each cell, time constants for individual responses were averaged, and these means then used to determine mean time constants for the populations of cells. Concentration-response curves were fitted using Prism software (Graphpad Software, San Diego, CA) using the equation (I = Imax/1 + 10(LogEC50-Log[17alpha -meT]n) where Imax equals the current observed with 1 mM GABA alone. Data from both acutely isolated cells and slice recordings were digitized at 23.6 kHz and filtered at 4 kHz for analysis.

Statistical analysis

Values given are means ± SE. Statistical significance was determined using Kolmogorov-Smirnov test for cumulative distributions, paired and unpaired two-tailed Student's t-tests for assessment of means, and by two-way ANOVA for analysis of concentration response curve fits. All statistical analyses were performed on non- transformed data.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GABAergic spontaneous inhibitory synaptic currents in the VMN and the mPOA

Spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in the presence of glutamatergic antagonists (see METHODS). Responses were reversibly blocked by 10 µM bicuculline (data not shown), and >50 sIPSCs were averaged per each cell and each experimental condition. Peak current amplitudes of sIPSCs from VMN neurons (109.7 ± 6.7 pA) were comparable with those elicited from mPOA neurons (119.8 ± 10.1 pA). Data collected in the present study were consistent with previous reports (Nett et al. 1999; Smith et al. 1996), indicating that >90% of individual sIPSCs from the VMN and the mPOA decayed with biexponential kinetics described by two time constants, tau 1 and tau 2 (Table 1). A third component of current decay (tau 3 >100 ms) was rarely observed and not included in the data analysis. In addition, the kinetics of current decay for sIPSCs were not significantly different from those estimated for miniature IPSCs (mIPSCs; data not shown). All responses were included in the analysis of sIPSCs with the exception of those with inflections on the rising phase, indicative of asynchronous multiple events, and those events with rise times >2 ms, which may have been distorted due to cable filtering. Such excluded events accounted for <10% of those acquired.


                              
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Table 1. Effects of 17alpha -meT on the kinetics of GABAA receptor-mediated currents

AAS effects on sIPSCs in the VMN and the mPOA

Greater than 60 AAS have been reported to be available on the United States market (including both the generic and the black markets) (Kammerer 1993). While 17alpha -meT, stanozolol, and nandrolone represent three of the most commonly abused AAS (Kammerer 1993), the chemical structures of these compounds represent two structurally distinct groups, the 17alpha -alkylated derivatives (17alpha -meT and stanozolol) and the 19-nortestosterone derivatives (nandrolone). In addition to being chemically distinct from one another, all three AAS have important structural differences with the neurosteroids. In particular, active neurosteroids have been reported to possess a 5alpha - or 5beta -reduced steroid ring skeleton with an alpha -hydroxyl at C3 and a keto group at either C20 in the pregnane ring or C17 in the androstane ring (Lambert et al. 1995), structural features that are not found in any of the three AAS examined here (Fig. 1).



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Fig. 1. Neurosteroid and anabolic-androgen steroid (AAS) structures. Chemical structures of the 2 endogenous neuroactive steroids (A) and the 3 AAS (B) shown to induce allosteric modulation of GABAA receptors in the ventromedial nucleus of the hypothalamus (VMN) and the medial preoptic area (mPOA).

To determine if AAS could modulate GABAA receptor-mediated synaptic transmission, neurons in the mPOA and the VMN 1 µM 17alpha -meT, stanozolol, or nandrolone, a concentration chosen to approximate that achieved in athletes abusing AAS (Wu 1997), significantly enhanced the average peak current amplitude of sIPSCs recorded from neurons of the VMN and significantly decreased the peak amplitude of sIPSCs recorded from neurons of the mPOA (Figs. 2 and 6). The fractions of responsive cells in the VMN were 14/14 (17alpha -meT), 12/15 (stanozolol), and 12/14 (nandrolone) and in the mPOA were 11/14 (17alpha -meT), 13/16 (stanozolol), and 12/14 (nandrolone). Response properties were stable during the data-acquisition periods prior to, during, and on washout of AAS, and the effects of each AAS were reversible (Fig. 2B). In the VMN, 1 µM 17alpha -meT also acted to enhance GABAergic sIPSCs by significantly increasing the values of both tau 1 and tau 2 without significantly altering the percentage of the peak current attributable to the fast component (%tau 1; Table 1) thus prolonging synaptic current decay in this region. As with 17alpha -meT, application of 1 µM stanozolol or nandrolone significantly increased the values of both tau 1 and tau 2 in the VMN with no change in %tau 1 (data not shown). In contrast to the VMN, no significant changes in current kinetics of sIPSCs for neurons of the mPOA were induced by application of 1 µM 17alpha -meT (Table 1) or stanozolol (data not shown). In agreement with results produced by these two 17alpha -alkylated AAS, no significant changes in the values of tau 1 and tau 2 for mPOA neurons were induced by nandrolone. However, this 19-nortestosterone derivative did significantly (P < 0.05) decrease %tau 1 for sIPSCs in the mPOA (data not shown). Because the slowest component of current decay (tau 3) was only rarely observed in recordings of sIPSCs, the effects of AAS on this kinetic component of synaptic responses could not be determined. Finally, AAS did not alter significantly the frequency of sIPSCs in either region (data not shown), suggesting the mechanism of action of these compounds is postsynaptic.



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Fig. 2. Modulation of spontaneous inhibitory postsynaptic currents (sIPSCs) by AAS. A: representative sIPSCs recorded from neurons of the VMN or the mPOA during perfusion with artificial cerebrospinal fluid (ACSF) or with ACSF containing 1 µM 17alpha -methyltestosterone (17alpha -meT), stanozolol, or nandrolone. Scale bars: 20 pA; 20 ms. Return to ACSF resulted in a return to baseline current levels (not shown). B: representative plot of all events from a VMN neuron exposed to 17alpha -meT illustrating the averaged peak amplitude of events recorded prior to AAS exposure (pre), during AAS exposure (+17alpha -meT), and following washout of the steroid (wash), as indicated by the 2 vertical, dashed lines. Response amplitudes were reversibly altered by steroid exposure and stable during each experimental condition. Individual events were acquired, fitted, and subsequently binned in 10-s increments where each data point represents the mean ± SE for the sIPSCs during that 10-s interval. Responses subsequently analyzed to determine AAS effects are indicated by the thick black bars. The slash on the time axis indicates a 5-min break in the record after changing from +17alpha -meT back to ACSF alone. Amplitude vs. time distributions of sIPSCs indicated that responses from all neurons from both regions accepted for analysis showed similar stability of recording and reversibility of AAS effects for stanozolol and nandrolone (data not shown) as well as for 17alpha -meT. C: cumulative probability histograms of the sIPSCs recorded from a VMN neuron (left) and an mPOA neuron (right) indicating that 17alpha -meT induced significant (P < 0.001) and proportional changes in peak amplitudes for all detected events. Stanozolol and nandrolone induced shifts comparable to that produced by 17alpha -meT for neurons from both regions (data not shown). Data shown in B and C were taken from the same recordings as shown for 17alpha -meT in A.

Responses elicited by ultrafast perfusion of GABA to isolated neurons of the VMN and the mPOA

While analysis of synaptic responses in acutely isolated slices provides the most physiologically relevant system to assess how AAS may alter synaptic transmission in the brain, concentrations of modulators that reach synapses within the slice may be appreciably less than those in the external solution (Brussaard et al. 1997). To determine the concentration-response relationships for AAS modulation of GABAA receptor-mediated currents, experiments were also performed on neurons acutely isolated from the VMN or the mPOA and exposed to GABA in the absence or presence of the 17alpha -meT using ultrafast perfusion techniques. GABAA receptor-mediated responses (Fig. 3A) were elicited by brief pulses (3 ms) of 1 mM GABA, application parameters believed to approximate those in the synaptic cleft (Jones and Westbrook 1995). Acutely isolated neurons from these postnatal animals did not adhere tightly to tissue culture substrates, precluding formation of outside-out patches from these cells with an acceptable success rate. However, the neurons used for these studies were small (Cavg = 5.1 ± 0.2 pF for VMN neurons; n = 164) and 3.8 ± 0.5 pF for mPOA neurons; n = 152) with estimated diameters between 5 and 10 µm, values comparable with those reported for nucleated outside-out patches from embryonic mouse forebrain neurons (Sather et al. 1992). Responses elicited by brief pulses of 1 mM GABA were evident in nearly all cells (>90%) and rose rapidly (10-90% rise times <= 2 ms), consistent with previous reports (Nett et al. 1999; Smith et al. 1996). Peak current densities (Ipeak) elicited by 1 mM GABA from VMN neurons were comparable (648.31 ± 71.66 pA/pF) with those elicited from mPOA neurons (635.76 ± 68.64 pA/pF), and as previously shown (Nett et al. 1999; Smith et al. 1996), the responses in neurons from both regions were in most cases best fitted by three kinetic components (Fig. 3A; Table 1). Specifically, for neurons from the VMN (n = 129), current decays for 50% of the responses were best fitted by three components. For neurons from the mPOA (n = 112), current decays for 71% of the responses were best fitted by three components. For both regions, in those neurons where current decays were best fitted by two components, it was the fastest component (tau 1) that was not observed.



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Fig. 3. Characteristics of responses elicited by ultrafast perfusion of 17alpha -meT. A: responses elicited from acutely isolated forebrain neurons by 3-ms pulses of 1 mM GABA. Representative response illustrating that current decays were best fitted by 3 exponential components indicated by the solid lines and described by the time constants tau 1, tau 2, and tau 3 (left). Numbers in parentheses indicate the percentage of the total peak current attributed to each kinetic component. Representative responses illustrating that coapplication of the AAS, 17alpha -meT reversibly enhanced currents elicited by 1 mM GABA from neurons isolated from the VMN (middle) but reversibly diminished responses from neurons of the mPOA (right). Scale bars: 200 pA; 100 ms. B: responses elicited by 3-ms pulses of 1 mM GABA from human embryonic kidney (HEK) 293 cells transiently transfected with cDNAs encoding the alpha 2, beta 3, and gamma 2 l or gamma 1 subunit and the green fluorescent protein. As with native neurons, current decays were best fitted by 3 exponential components (left) and co-application of 17alpha -meT reversibly enhanced currents elicited from cells transfected with alpha 2, beta 3, and gamma 2 l cDNAs (middle) while reversibly diminishing currents from cells transfected with alpha 2, beta 3, and gamma 1 cDNAs (left). Scale bars: 500 pA; 100 ms.

AAS modulation of responses elicited by ultrafast perfusion of GABA to isolated neurons of the VMN and the mPOA

The AAS, 17alpha -meT, modulated GABAA receptor-mediated currents from isolated VMN and mPOA neurons in a manner consistent with its effects on sIPSCs in the two brain regions. As with sIPSCs, coapplication of 1 µM 17alpha -meT with 1 mM GABA significantly increased Ipeak (Figs. 3A and 6) and the value of tau 1 (Table 1) for neurons of the VMN, but decreased Ipeak (Figs. 3A and 6) with no change in tau 1 or tau 2 (Table 1) for neurons of the mPOA. Modulation by 17alpha -meT was elicited in the absence of preequilibration with the steroid, supporting the assertion that the AAS act at an extracellular allosteric site on the receptor. For currents elicited by ultrafast perfusion of GABA to isolated mPOA neurons, 1 µM 17alpha -meT also significantly increased the value of tau 3 (Table 1), a kinetic component of current decay rarely observed in synaptic responses. Application of 0.01% DMSO alone, the carrier for AAS, did not elicit responses or modulate GABAA receptor-mediated currents (data not shown, n = 5).

While millimolar concentrations of GABA are believed to reflect those in the synaptic cleft, tonic activation of both synaptic and extrasynaptic GABAA receptors (Brickley et al. 1996) may occur at significantly lower concentrations. Therefore the effects of 1 µM 17alpha -meT were also assessed for responses elicited by brief (3 ms) pulses of 10-6 to 10-2 M GABA (Fig. 4A). Measurable responses were not evident with concentrations of GABA <= 5 µM, consistent with previous reports for alpha 2-containing receptors (Lavoie and Twyman 1996), and maximal currents were elicited by 10 mM GABA in both regions. EC50 values for GABA were 32 µM for the VMN and 46 µM for the mPOA, and the Hill coefficient was ~1 for neurons from both regions. Coapplication of 1 µM 17alpha -meT altered the efficacy of GABA, augmenting Ipeak in VMN neurons and diminishing Ipeak in mPOA neurons for concentrations of GABA >= 10 µM (Fig. 4A). Similar concentration-response relationships were obtained for the prolongation of current decay kinetics by 17alpha -meT in the VMN (data not shown). The EC50 values for GABA were shifted in response to coapplication of 1 µM 17alpha -meT to 11 µM in the VMN and 19 µM in the mPOA; however, these shifts were not significant.



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Fig. 4. Dose-dependent effects of AAS on brief pulses of GABA. A: concentration-response relationships of average Ipeak constructed from responses of neurons first exposed to concentrations of GABA from 1 µM to 10 mM () and subsequently to GABA plus 1 µM 17alpha -meT (black-triangle) for the VMN (left: n = 5-9 cells for each data point) and the mPOA (right: n = 4-8 cells for each data point). Error bars indicate SE; *, significance in comparison of mean AAS values with control (*0.01 < P < 0.05; **0.01 > P > 0.001; ***P < 0.001). B: concentration-response relationships were from neurons exposed to 1 mM GABA plus 10 nM to 50 µM 17alpha -meT. In the VMN (left), 17alpha -meT significantly enhanced Ipeak at 1 µM and 10 µM. No potentiation was observed in the VMN with 50 µM 17alpha -meT, and this point was omitted from the fit. The enhancement of current decay by 17alpha -meT in the VMN was similarly dose-dependent (data not shown); n = 17-22 cells for each data point. In the mPOA (right), Ipeak was decreased significantly by 17alpha -meT at 1, 10, and 50 µM; n = 15-23 cells for each data point.

To establish the concentration range of AAS required to induce significant changes of GABAA receptor-mediated responses under conditions that mimic synaptic transmission, concentration response relationships were also determined for responses elicited by brief (3 ms) applications of 1 mM GABA and 10-8 to 10-4 M 17alpha -meT (Fig. 4B). For VMN neurons, significant potentiation of Ipeak was observed with 10-6 and 10-5 M 17alpha -meT. Potentiation was not observed for responses from VMN neurons with 10-4 M 17alpha -meT. For mPOA neurons, Ipeak was significantly decreased by 10-6 to 10-4 M 17alpha -meT (Fig. 4B). Assessment of concentration response relationships for neurons from both regions indicated that half-maximal effects on Ipeak (Fig. 4B) and decay kinetics (data not shown) were achieved by concentrations of 17alpha -meT in the submicromolar range: EC50 = 238 nM for the VMN and IC50 = 857 nM for the mPOA. Application of 17alpha -meT alone at concentrations between 1-50 µM did not elicit responses in the absence of GABA (n = 10; data not shown).

AAS modulation of recombinant receptors expressed in HEK293 cells

Previous studies have indicated that the predominant isoform of GABAA receptors expressed in the VMN is alpha 2beta 3gamma 2 (Wisden et al. 1992) while the receptor type that predominates in the mPOA is alpha 2beta 3gamma 1 (Herbison and Fénelon 1995; Wisden et al. 1992). Inclusion of a gamma 1 subunit has been shown to confer unusual pharmacology for both native (Bormann and Kettenmann 1988; Nett et al. 1999) and recombinant GABAA receptors (Puia et al. 1991; Wafford et al. 1993). To test if the opposing modulation elicited by AAS in neurons of the VMN versus the mPOA arises from region-specific expression of gamma 2- versus gamma 1-containing receptors in the VMN and the mPOA, respectively, we assessed modulation by 17alpha -meT of GABAergic currents elicited from HEK293 cells transiently transfected with cDNAs encoding the green fluorescence protein (GFP) and either cDNAs encoding the alpha 2, beta 3, and gamma 2 or the alpha 2, beta 3, and gamma 1 receptor subunits. Previous studies have shown that >90% of cells expressing GFP also expressed GABAA receptors (Zhu et al. 1996), a result confirmed in the present study. Recordings were made from transfected HEK293 cells that were small in size (<5 pF) and not coupled to other cells. Currents elicited by a 3-ms pulse of 1 mM GABA elicited currents with Ipeak = 159 ± 47.4 pA/pF for cells transfected with the alpha 2, beta 3, and gamma 2 l cDNAs (n = 5) and 323.6 pA ± 51.5 pA/pF for cells transfected with the alpha 2, beta 3, and gamma 1 cDNAs (n = 7). As with native neurons, current decays of GABAA receptor-mediated responses from HEK293 cells were described by three time constants as were currents from native receptors. Decay time constants were tau 1 = 3.80 ± 0.39 ms, tau 2 = 67.58 ± 12.54 ms, and tau 3 = 731.8 ± 211.7 ms for cells transfected with the alpha 2, beta 3, and gamma 2 l receptor subunit cDNAs. Time constants for cells transfected with the alpha 2, beta 3, and gamma 1 receptor subunit cDNAs were 7.37 ± 2.07, 38.15 ± 10.25, and 228 ± 20.6 ms, respectively, for tau 1, tau 2, and tau 3. Coassembly of a gamma  subunit is known to be required for benzodiazepine sensitivity of GABAA receptors (for discussion, Sieghart 1995). Consistent with inclusion of gamma , as well as alpha , and beta  subunits in recombinant receptors, the benzodiazepine, diazepam, was found to reversibly potentiate Ipeak from both sets of transfected cells (n = 6; data not shown). Most important, as with currents elicited from neurons of the VMN, 17alpha -meT significantly (P < 0.03; n = 6) and reversibly enhanced currents from cells transfected with the alpha 2, beta 3, and gamma 2 receptor cDNAs (Figs. 3B and 6). Conversely, 17alpha -meT significantly (P < 0.03; n = 5) and reversibly decreased currents from cells transfected with the alpha 2, beta 3, and gamma 1 receptor cDNAs (Figs. 3B and 6), an effect that mirrors the modulation by this AAS for neurons from the mPOA. The magnitude of negative modulation of Ipeak induced in HEK cells transfected with cDNAs encoding the alpha 2, beta 3, and gamma 1 receptor subunits was not significantly different from that observed for currents elicited by ultrafast perfusion of mPOA neurons (Fig. 6). In contrast, while 17alpha -meT enhanced current densities for both VMN neurons and HEK cells transfected with cDNAs encoding the alpha 2, beta 3, and gamma 2 l receptor subunits, the magnitude of the increase was significantly higher (P < 0.001) for the transfected cells (Fig. 6). Taken together with data from VMN and mPOA neurons, the data from analysis of responses elicited by ultrafast perfusion of GABA to transiently transfected HEK293 cells indicate that the opposing pattern of modulation by AAS can be attributed, at least in part, to the preferential expression of gamma 1-containing neurons in the mPOA and of gamma 2-containing neurons in the VMN.

Neurosteroid effects on spontaneous inhibitory synaptic currents in the VMN and the mPOA

While allosteric modulation of GABAA receptors by endogenous neuroactive steroids is well-characterized (for review, Lambert et al. 1995), critical differences in chemical structure between the AAS and the endogenous neuroactive steroids raised the possibility that these steroid compounds may have different mechanisms of interacting with the GABAA receptor. To determine if this was the case, we also assessed the ability of two endogenous neuroactive steroids, the progesterone derivative, 3alpha ,5alpha -THP, and the testosterone derivative, 3alpha -DIOL, to modulate sIPSCs in neurons in the VMN and the mPOA. Both 3alpha ,5alpha -THP and 3alpha -DIOL potentiated sIPSCs in the VMN (3alpha ,5alpha -THP: 5/5 cells; 3alpha -DIOL: 7/10 cells; Figs. 5 and 6). However, in contrast to the diminution produced by AAS in the mPOA, both of these neuroactive steroids significantly enhanced sIPSCs in the mPOA (3alpha ,5alpha -THP: 6/10 cells; 3alpha -DIOL: 7/11 cells; Fig. 5, A and B, and 6). Testosterone itself, was without effect (Fig. 5B; n = 5). Enhancement of current amplitudes by endogenous neuroactive steroids and diminution by AAS was observed in the same neuron in the mPOA (Fig. 5B), indicating that the differences in responses to AAS and neurosteroids were not attributable to selective subpopulations of neurons within this region. These data show that synaptic transmission in forebrain neurons of the VMN and the mPOA can be modulated by both endogenous neuroactive steroids and AAS but that the modulation induced by the two classes of steroids for neurons of the mPOA is not equivalent.



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Fig. 5. Modulation of sIPSCs by endogenous neuroactive steroids. A: representative sIPSCs recorded from neurons of the VMN or the mPOA during perfusion with ACSF and with ACSF containing 1 µM of the testosterone derivative, 5alpha -androstane-3alpha ,17beta -diol (3alpha -DIOL), or the progesterone derivative, 3alpha -hydroxy-5alpha -pregnan-20-one (3alpha ,5alpha -THP), indicating that both neuroactive steroids increased sIPSC amplitudes in neurons from both regions. The enhancement was reversible (not shown). B: representative sIPSCs recorded from an mPOA neuron in ACSF and in ACSF plus 1 µM testosterone showing no effect of the parent steroid (left). Representative sIPSCs illustrating that sequential application of 1 µM 3alpha -DIOL, followed by return to control (not shown) and then 1 µM 17alpha -meT in the same mPOA neuron induced first enhancement then diminution of peak current amplitude (right). Scale bars: 20 pA; 20 ms.

Comparison of steroid effects on GABAA receptor-mediated currents

Taken together, our results indicate that all three AAS induced rapid modulation of GABAA receptor-mediated currents but that the pattern of AAS modulation differs for neurons of the VMN versus the mPOA (Fig. 6). Both the pattern and the extent of modulation in native neurons and HEK cells is consistent with alpha 2beta 3gamma 1 receptors predominating in neurons of the mPOA. However, differences in the percent of potentiation between VMN neurons and HEK cells expressing gamma 2-containing receptors (Fig. 6), as well as previous assessments of subunit mRNA (Wisden et al. 1992) and protein levels (Fritschy and Mohler 1995), suggest that receptor subtypes other than alpha 2beta 3gamma 2 l are also expressed at appreciable levels in this nucleus and may play a role in determining the sensitivity of VMN neurons to AAS modulation. Finally, our results indicate that endogenous neuroactive steroids also modulate GABAA receptor-mediated currents in neurons from the VMN and the mPOA but enhance rather than diminish responses in the mPOA, which is in contrast to AAS in this region (Fig. 6). These data suggest that the AAS may have different mechanisms for altering GABAA receptor function than do the endogenous neuroactive steroids.



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Fig. 6. Comparison of steroid modulation of peak currents. Cumulative histograms for data acquired from the VMN and HEK293 cells transfected with alpha 2beta 3gamma 2 cDNAs (A) and the mPOA and HEK293 cells transfected with alpha 2beta 3gamma 1 cDNAs (B), indicating the relative enhancement or diminution of peak current amplitudes induced by 1-µM concentrations of both the endogenous neuroactive steroids, 3alpha -DIOL and 3alpha ,5alpha -THP, and the AAS, 17alpha -meT, stanozolol, and nandrolone. Modulation is illustrated for synaptic currents from neurons in brain slices (sIPSCs) and for currents elicited by ultrafast perfusion of 1 mM GABA (3 ms) and 1 µM 17alpha -meT to acutely isolated cells (perfusion), either native neurons of the VMN or the mPOA or transiently transfected HEK293 cells (HEK) expressing recombinant receptors. Control values are indicated by 100%. Error bars indicate SE; *, significance in comparison of mean AAS values with control (*0.01 < P < 0.05; **0.01 > P > 0.001; ***P < 0.001).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Here we show for the first time that three commonly abused AAS, 17alpha -meT, stanozolol, and nandrolone, can induce rapid and reversible modulation of GABAA receptor-mediated synaptic transmission in neurons from the mammalian brain. These data provide direct evidence that the AAS join a long list of allosteric modulators of the GABAA receptor and suggest a cellular mechanism by which these compounds may alter CNS function. We found that peak current amplitudes and decay kinetics of sIPSCs were enhanced by these three AAS in all responsive neurons of the VMN. In contrast, peak current amplitudes of sIPSCs were decreased, without concomitant changes in decay kinetics (with the exception of a decrease in %tau 1 induced by nandrolone), in all responsive neurons from the mPOA. No significant differences were induced in the frequency of sIPSCs on application of AAS, suggesting the primary site of action of these compounds is postsynaptic, a conclusion further supported by the ability of 17alpha -meT to modulate GABA responses of isolated neurons. While the endogenous androgen metabolite, 3alpha -DIOL, also induced reversible modulation of GABAA receptor-mediated sIPSCs, application of testosterone did not, indicating that the effects of these androgens were not due to nonspecific membrane effects of steroids. Results indicating that ultrafast perfusion of 1-50 µM 17alpha -meT in the absence of GABA to dissociated cells had no effect similarly argue against nonspecific effects of these steroids.

To obtain concentration response data for AAS under conditions where both the concentration of GABA and the concentration of the AAS could be accurately controlled, currents were also elicited from acutely isolated neurons of the VMN and the mPOA by ultrafast perfusion of GABA in the absence or presence of AAS. Initial assessment of a single concentration (1 µM) of 17alpha -meT on responses elicited by brief (3 ms) pulses of 1 mM GABA indicated that this AAS modulated GABAA receptor-mediated responses from isolated cells in a manner comparable with that observed for synaptic responses in intact slices. Specifically, 17alpha -meT significantly enhanced peak current amplitudes and the value of tau 1 for neurons from the VMN but diminished peak current amplitudes with no change in tau 1 or tau 2 for neurons of the mPOA. For isolated neurons from the mPOA, 17alpha -meT also induce a significant prolongation of tau 3 of responses elicited by ultrafast perfusion of GABA. The low frequency of occurrence of this component in synaptic responses precluded a comparison of AAS effects on this parameter between the two experimental paradigms, and the relevance of this component in currents elicited by ultrafast perfusion remains unclear. It is possible that there are limitations in solution exchange time with direct perfusion techniques that arise from the use of the whole cell configuration (Jonas 1995), even with the small neurons used in this study, and although this component was consistently observed, it may not reflect a physiological process that is relevant to synaptic transmission in the brain. Conversely, this slow component of decay may also arise from extrasynaptic receptors that would not be activated under conditions of low, spontaneous synaptic activity but may contribute to physiologically relevant GABAA receptor-mediated responses in intact slices under conditions of high-frequency release and spillover to extrasynaptic receptors (Chéry and DeKoninck 1999; Rossi and Hamann 1998). Concentration response relationships indicated that 1 µM 17alpha -meT significantly altered the efficacy of a broad range of GABA concentrations (10 µM to 10 mM), suggesting that modulation by AAS will occur in situ for both saturating (Jones and Westbrook 1995) and subsaturating (Frerking et al. 1995; Hill et al. 1998; Nusser et al. 1997) conditions at neuronal GABAA receptors. AAS may therefore act to influence not only discrete GABAergic IPSCs but also the tonic actions of GABA that have been shown influence electrical excitability (Brickley et al. 1996).

In the VMN, our data indicate that both AAS and endogenous neurosteroids potentiated sIPSC amplitudes as well as prolonged the slower time constant (tau 2) of synaptic current decay. The observed enhancement of sIPSC amplitude, when taken in conjunction with data from fast perfusion experiments demonstrating that concentrations as high as 10 mM GABA were required to elicit maximal responses from these neurons, suggest GABA concentrations at synapses in the VMN may be subsaturating as has been reported for other brain regions (Frerking et al. 1995; Hill et al. 1998; Nusser et al. 1997). Conversely, although we did not include responses that were notably asynchronous and multiquantal in our analysis, small inflections in the rising phase may have not been detected and thus some degree of potentiation of sIPSCs by steroids may also reflect the integration of multiple mIPSCs whose durations are prolonged by AAS (Mody et al. 1994).

With respect to delineating the concentrations of AAS that induce significant modulation of GABAergic responses, we found that half-maximal effects of 17alpha -meT were in the hundred nanomolar range in both the VMN and mPOA. While the physiological range of circulating endogenous androgens in women has been determined to be appreciably below this level (2 nM) (Wu 1997), plasma concentrations of AAS in steroid abusers have been estimated to be 100- to 1,000-fold higher than those of these endogenous androgens (Wu 1997). Therefore concentrations of AAS that produced significant modulation of GABAergic currents are likely to be reached in individuals who abuse these drugs.

GABAA receptors are expressed throughout the mammalian brain; however, there is a broad range of structural heterogeneity that in turn gives rise to a plethora of region-specific differences in GABAA receptor function (for review, Sieghart 1995). Our results demonstrate that although the AAS produce significant modulation of GABAergic currents in both the VMN and the mPOA, these steroids induce an opposing pattern of modulation in these two brain regions. In conjunction with previous studies indicating that the VMN and the mPOA differ dramatically in the expression of gamma  subunit isoforms (Clark et al. 1998b; Herbison and Fénelon 1995; Wisden et al. 1992; Ymer et al. 1990), the data presented here from analysis of recombinant receptors expressed in heterologous cells strongly suggest that the preferential expression in the mPOA of receptors containing gamma 1 subunits underlies the negative modulation of currents in neurons of this region. The expression of gamma 1-containing receptors is restricted to a handful of brain regions involved with the production and affective components of reproductive behaviors (for discussion, see Nett et al. 1999), suggesting that the negative allosteric modulation induced by AAS may have unique actions on neuroendocrine functions. While the data from all experiments performed here are consistent with the predominant expression of gamma 2-containing receptors underlying the positive modulation by AAS of neurons in the VMN, 17alpha -meT induced significantly greater potentiation of responses from HEK cells transfected with alpha 2, beta 3, and gamma 2 subunit cDNAs than from VMN neurons. These data suggest that receptors other than alpha 2beta 3gamma 2 combination may contribute to AAS sensitivity. In particular, the role of alpha 5 (Fritschy and Mohler 1995; Wisden et al. 1992) and epsilon  subunits (Davies et al. 1997; Whiting et al. 1997) should be explored since these receptor subunits are expressed at high levels in the VMN and have been implicated in determining the actions of other classes of allosteric modulators (Davies et al. 1997; Whiting et al. 1997; for review, Sieghart 1995). In addition, our data suggest that receptor heterogeneity will need to be considered as an important determinant of how AAS alter GABAA receptor function in brain regions that play a significant role in mediating aggression and anxiety, behaviors affected by AAS in both rodents (Bitran et al. 1993; Bronson 1996; Bronson et al. 1996) and in human abusers (Moss et al. 1992; Pope and Katz 1988).

There is a wealth of data demonstrating that endogenous neuroactive steroids act as allosteric modulators of GABAA receptors (for review, Lambert et al. 1995) and that these compounds have significant effects on neuroendocrine behaviors (for review, Majewska 1987). The results presented here, however, do not support the assertion that the AAS act equivalently to the endogenous neuroactive steroids in the CNS. Specifically, the pattern of modulation induced by AAS in the mPOA was significantly different from that observed with exposure to the endogenous neuroactive steroids, 3alpha ,5alpha -THP and 3alpha -DIOL. All three AAS induced negative modulation of mean current amplitude in the mPOA, while 3alpha ,5alpha -THP and 3alpha -DIOL enhanced the average peak current in this region. Moreover, no responses were elicited by concentrations of AAS as high as 50 µM in the absence of GABA, whereas high concentrations of neurosteroids are known to activate GABAA receptors directly (for review, Lambert et al. 1995). In fact, the characteristics of AAS modulation of GABAA receptors in mPOA neurons are more reminiscent of those reported for the benzodiazepine site modulator, zolpidem, (Nett et al. 1999) than to those elicited by these endogenous steroids. For example, not only do AAS induce negative modulation in mPOA neurons as does zolpidem, but the dose-response curve for VMN neurons is bell-shaped as has been reported previously for both positive and negative modulators acting at the benzodiazepine site (Rovira and Ben-Ari 1993; Sigel et al. 1990; Stevenson et al. 1995). It is noteworthy that key structural elements shown to be common to all active neurosteroids (Fig. 1) (for review, Lambert et al. 1995) are absent from the three AAS tested here. While the precise mechanisms by which AAS alter GABAA receptor function remain to be determined, our results indicate that AAS may mirror the actions of other modulators, such as the benzodiazepines, more closely than those produced by the endogenous neuroactive steroids.

Analysis of AAS and neurosteroid effects on sIPSCs in both the VMN and the mPOA were performed on animals that were an average age of PN13 (range PN10-PN17). Developmental changes in GABAA receptor subunit composition in the hippocampus have been shown to result in significant decreases in the sensitivity of granule cells to the endogenous neurosteroid, 3alpha , 21 dihydroxy-5alpha -pregnan-20-one (THDOC), between PN10-PN20; a change that may arise from the late developmental expression of the delta  subunit (Cooper et al. 1999; Zhu et al. 1996). Previous studies have demonstrated that expression of mRNAs encoding the alpha l and alpha 2 subunits (Davis et al., 2000) as well as the beta  subunits (Zhang et al. 1991) undergo developmental changes in expression in the VMN and the mPOA during this postnatal period. Moreover, unpublished data from our laboratory (A. S. Clark, S. Robinson, and L. P. Henderson) indicate that there are significant changes in expression of the gamma 1 subunit in the mPOA during the first two postnatal weeks. Both alpha  and gamma  subunits have been shown to influence the sensitivity of recombinant receptors to 3alpha ,5alpha -THP (Lambert et al. 1999; Maitra and Reynolds 1998; Puia et al. 1993), and gamma  subunit expression, as we have shown here, has significant effects on AAS modulation. Assessment of the percent modulation of sIPSCs as a function of development for the data in this study suggests that the magnitude of modulation induced in the VMN and the mPOA by both the AAS and the neurosteroids may change over this epoch. However, a conclusive timeline for developmental changes in the sensitivity of neurons in these regions to steroid modulation will require a more comprehensive study. In particular, it will be of interest to determine if the sensitivity to either class of these steroid modulators changes not only during the period of active synaptogenesis within the first few weeks of development, but also if there are subsequent changes concomitant with the onset of puberty.

The ubiquitous expression of GABAA receptors throughout the brain suggests that AAS will have widespread effects on a broad range of CNS functions; however, the data presented here have particular relevance with regard to delineating mechanisms by which AAS may alter reproductive behaviors. For example, GABAergic transmission in both the mPOA and the VMN regulates the expression of sexual receptivity in female rodents (for review, Pfaff et al. 1994), and neurosteroid modulators of GABAA receptors (for review, McCarthy 1995), including the endogenous androgen, 3alpha -DIOL, (Frye et al. 1996) facilitate female sexual behaviors when acutely infused into the VMN and its efferent targets. Although chronic AAS exposure may modify sexual behaviors via a number of signaling pathways, including actions mediated by nuclear androgen receptors (Blasberg et al. 1998), our data suggest that the AAS may also have acute effects on sexual behaviors, as do the endogenous neuroactive steroids, via actions at the GABAA receptor. While extrapolation from receptivity in rats to sexual behaviors in human abusers must be made with caution, cellular actions of AAS at the GABAA receptor may underlie some of the reported changes in sexual performance and libido in steroid abusers (Franke and Berendonk 1997). In addition, GABAergic control of gonadotropin-releasing hormone (GnRH) pulsatility by neurons of the mPOA is essential both for the onset of puberty (for review, Ojeda and Urbanski 1994) and for establishing estrous cyclicity in adult females (for review, Freeman 1994). AAS interactions with GABAergic control neurons of the mPOA may contribute to the changes in gonadotropin secretion (Bronson et al. 1996), irregular cyclicity (Blasberg et al. 1997; Bronson 1996; Bronson et al. 1996; Clark et al. 1998a), and accelerated reproductive senescence (Bronson 1996) reported with AAS use.

Although few studies have been carried out on either women or female rodents, the available data suggest that females are more sensitive to the actions of AAS than males (Bronson 1996; Hickson and Kurowski 1986). Previous studies have demonstrated that AAS effects on benzodiazepine binding to GABAA receptors are not equivalent in female and male rats (Masonis and McCarthy 1995), suggesting that actions of these steroids at the GABAA receptor are also sexually dimorphic. Given that the most dramatic increases in AAS abuse are among women and especially young girls (Bahrke et al. 1998), defining how these steroids act in both sexes, as well as determining if they have significantly different actions in prepubertal versus adult subjects, will be essential for understanding the adverse effects of these steroids in the CNS and the implications their use has for reproductive health.


    ACKNOWLEDGMENTS

We thank Dr. Stefano Vicini for providing us with GABAA receptor subunit cDNAs and Drs. Ann Clark and Anita Prasad for a critical review of the manuscript.

This research was supported by the National Institute of Neurological Disorders and Stroke (NS-28668 to L. P. Henderson) and the National Science Foundation (DBI-9707826 to J. C. Jorge-Rivera).


    FOOTNOTES

Address for reprint requests: L. P. Henderson, Dept. of Physiology, Dartmouth Medical School, Hanover, NH 03755.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 December 1999; accepted in final form 9 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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