Introduction

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3,5-P and 5-THDOC are reduced metabolites of progesterone and deoxycorticosterone, respectively. They are among the most potent endogenously occurring neuroactive steroids with high specificity for the GRC (Gee et al., 1987, 1988; Harrison et al., 1987; Lan et al., 1990; Majewska et al., 1986; Morrow et al., 1987, 1990; Peters et al., 1988; Turner et al., 1989). In contrast to hormonal steroids, these neuroactive steroids activate a membrane-bound steroid site to exert rapid and reversible effects on GABA-gated Cl channel conductance. The remarkable pharmacological potency of these steroids and the unique structure-activity requirements of their receptors distinguish them from the hormonal steroids and their cytosolic receptors. Consistent with their GABA-agonist like activity at the GRC, these neuroactive steroids share similar pharmacological effects with benzodiazepines and barbiturates, including anesthetic, sedative-hypnotic, anxiolytic and anticonvulsant actions (Gee et al., 1995). 3,5-P and 5-THDOC allosterically modulate the GRC in a manner reminiscent of a ligand that potentiates GABA action. For example, these steroids inhibit [³⁵S]TBPS and potentiate [³H]muscimol and [³H]flunitrazepam binding (Gee et al., 1988; Majewska et al.; 1986).

Endogenously occurring neuroactive steroids have been reported to reach concentrations in the brain well within the range necessary to potentiate the actions of GABA (Gee et al., 1987; Majewska et al., 1986; Paul et al., 1991; Purdy et al., 1991). These findings have provided the impetus to determine the possible physiological role of these compounds. As a part of this effort, studies have revealed neuroactive steroids with a varying range of efficacies as modulators of the GRC (Gee et al., 1988; Gee and Lan, 1991). Neuroactive steroids with partial agonist and antagonist activity have been reported (Gee and Lan, 1991; McCauley et al., 1995). The former may have significant implications for the development of pharmacological agents with therapeutic value, whereas the latter are of interest as tools for evaluation of the physiological importance of endogenous neuroactive steroids. Previous studies with 5-THDOC and its stereoisomer 5-THDOC revealed important differences in potency, efficacy and regional selectivity at the GRC, accounted for by only a difference in the spatial orientation of the steroid A-ring (Gee and Lan, 1991). The chemical structures showing this difference in spatial orientation are depicted in figure 1. In contrast to the 5-reduced analogs, 5-THDOC has limited efficacy and, under certain conditions, antagonist actions at the GRC in vitro. This interesting pair of neuroactive steroids provided the means to evaluate in detail the site and mechanism of action of 5-THDOC in vitro and in vivo.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Chemical structures of 5- and 5-THDOC.

	Materials and Methods

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Tissue Preparation

The brains from male Sprague-Dawley rats (160-200 g; Simonsen Laboratories, Gilroy, CA) were removed immediately after sacrifice, and the frontal cortex from each animal was dissected over ice. A P₂ homogenate was prepared for radioligand binding assays as described previously (Gee et al., 1987). Briefly, the tissue was gently homogenized (with a Teflon pestle) as a 10% (w/v) suspension in 0.32 M sucrose, followed by centrifugation at 1000 × g for 10 min at 0° to 4°C. The supernatant was collected and centrifuged at 9000 × g for 20 min at 0° to 4°C. The resultant pellet was washed three times in 100 volumes of ice-cold PBS (50 mM sodium/potassium phosphate, 200 mM NaCl, pH 7.4) by centrifugation at 9000 × g for 10 min and resuspended as a 10% (w/v) homogenate for immediate use in binding assays.

Stable Cell Lines

Stable expression of GABA_A receptor subunits. Human alpha-1 and gamma-2L GABA_A receptor subunits were cloned into mammalian expression vectors containing the constitutive enhancer/promoter of the immediate early gene of human cytomegalovirus, pCDM8 (InVitrogen, San Diego), or pcDNA1 (InVitrogen). The human beta-2 cDNA was obtained from the rat beta-2 sequence (Ymer et al., 1989) through mutation of the codon for amino acid 347 from Asn to Ser (Hadingham et al., 1993) and then ligation into the pcDNA1 expression vector.

Membrane preparation. Cells were harvested by removing the incubation media and replacing with 1 ml of 10× trypsin-EDTA solution (Life Technologies). After 5-min incubation with gentle agitation, 9 ml of serum-containing media was added, and the cells were released from the flask by gentle pipetting up and down of the culture medium. The culture medium was then removed by low-speed centrifugation at 1000 × g for 10 min and rinsed twice with cold 200 mM NaCl/50 mM Na-K phosphate, pH 7.4, buffer (PBS). Cell membranes were disrupted using a Polytron (Brinkmann Instruments, Westbury, NY) at setting 10 for 20 sec. The cell homogenate was centrifuged at 9000 × g for 20 min, and the pellet rinsed once before resuspension with cold PBS in the desired volume for the [³⁵S]TBPS binding assay.

[³⁵S]TBPS binding assay. [³⁵S]TBPS (2 nM, 60-120 Ci/mmol; New England Nuclear, Boston, MA) was incubated with 100-µl aliquots of cortical P₂ homogenate or cell membrane containing GRC alpha-1-beta-2-gamma-2L subunits in the presence and absence of various concentrations of steroids. All test drugs were dissolved in DMSO (Sigma Chemical Co, St Louis) and added to the incubation mixture in 5-µl aliquots. The incubation mixture was brought to a final volume of 1 ml with assay buffer. Nonspecific binding was defined as binding in the presence of 2 µM TBPS (Research Biochemicals, Natick, NH). The binding assays were performed in the presence or absence of 5 µM GABA (IC₅₀ value for GABA inhibition of [³⁵S]TBPS binding under the condition used). The incubation (90 min, 25°C) was terminated by rapid filtration through glass fiber filters (No. 32; Schleicher & Schuell, Keene, NH). The filters were washed three times with 3 ml of ice-cold phosphate buffer, and filter-bound radioactivity was quantified by liquid scintillation spectrophotometry. Protein concentration was determined according to the method of Lowry et al. (1951). The dose-response data were evaluated by computerized nonlinear regression (InPlot; GraphPAD, San Diego, CA) using a one-component (three-parameter) model to generate IC₅₀ values (Boxenbaum et al., 1971). The data collected from the receptor binding assays were analyzed by ANOVA and Newman-Keuls (P < .05) when warranted (Winer, 1971).

Electrophysiological Studies

Preparation of cRNA. Preparation of cRNAs for the alpha-1, beta-2 and gamma-2L subunits was performed as described previously (Hadingham et al., 1993; Ishiura et al., 1982; Ymer et al., 1989). cRNA was diluted to 1 µg/µl with DEPC-treated water and stored in 1- to 2-µl aliquots at 80°C until injection. Stocks of cRNA were thawed, mixed and diluted as noted below in H₂O immediately before injection.

Xenopus laevis oocyte expression system. Mature female X. laevis (Xenopus I, Ann Arbor, MI) were immersed in 0.15% 3-aminobenzoic ethyl ester (MS-222; Sigma Chemical, St. Louis, MO) until fully anesthetized (30-45 min), and two to four ovarian lobes were surgically removed and placed into Barth's medium containing (in mM) 88 NaCl, 1 KCl, 0.41 CaCl₂, 0.33 Ca(NO₃)₂, 0.82 MgSO₄, 2.4 NaHCO₃ and 5 HEPES, pH adjusted to 7.4 with NaOH. With slight modifications of established procedures (Woodward et al., 1994), oocytes (developmental stages V and VI) were plucked from the ovary and enzymatically defolliculated by treatment for 45 to 60 min with collagenase (0.5 mg/mL, Boehringer-Mannheim, Indianapolis, IN). After brief vortex-mixing to dislodge epithelia, theca and most of the follicular layer, oocytes were rinsed extensively with fresh Barth's medium and incubated overnight in Barth's medium supplemented with gentamycin (0.1 mg/ml). Individual oocytes were microinjected with a 5:1:1 ratio of cRNA encoding the GABA_A receptor subunits alpha-1, beta-2 and gamma-2L (~5 ng of the alpha-1 subunit and ~1 ng each of beta-2 and gamma-2L/oocyte). After injection, oocytes were maintained in Barth's supplemented with gentamycin (0.1 mg/ml) at 15° to 18°C.

Drugs. Neurosteroids were dissolved at 10 mM in DMSO (Sigma) and further diluted to make a series of DMSO stock solutions over the range of 0.001 to 10 mM. Working solutions were made by dilution of these DMSO stock solutions into Ringer's solution just before application, with final DMSO concentrations of 0.1% to 0.3%. At this dilution, DMSO alone had little or no measurable effects on the GABA control responses. DMSO stocks were stored at room temperature in the dark for ~7 days without apparent changes in potency. The neurosteroids 3,5-P and 5-THDOC were synthesized by CoCensys, Inc. (Irvine, CA); other reagents were from Sigma.

Experimental design and data analysis. GABA concentration-response data were obtained through successive exposures to increasing concentrations of GABA, until an apparent maximal current was reached (3-10 mM GABA). These data were analyzed using a PC-based graphing program (Origin, Microcal, Inc.). The following logistic equation was used to fit individual concentration-response data, where n is the slope, EC₅₀ is the concentration of GABA that produces a half-maximal response, I is the current at a given concentration of GABA and I_max is the maximal current in response to GABA. For steroid modulation experiments, oocytes were repeatedly exposed to 3 to 10 mM GABA, which elicited maximal currents. Maximal GABA responses were then allowed to stabilize, and a concentration of GABA that evoked current ~5% of the maximum was determined. This was used as the GABA control response for modulation by steroids. The concentration of GABA required to elicit 5% control responses varied between 3.3 and 11 µM. Oocytes were then exposed to steroids for 30 to 60 sec before coapplication of steroid with the control GABA solution. Fractional currents resulting from steroid alone or coapplication of steroid with GABA were obtained by dividing the current by the GABA maximal current. (In some cases, a sliding scale was used to account for changes in GABA maxima during the course of a single experiment.) These values were analyzed as above using the following equation to obtain EC₅₀ and slope values for steroid effects. For this equation, I_max is the GABA maximal current, I is the current obtained from steroid in combination with GABA, n is the slope and EC₅₀ is the concentration of steroid that produces half-maximal potentiation. All numerical values in the text represent mean ± S.E.M.

Behavioral studies. Male Swiss Webster mice (23-28 g, Simonsen Laboratories, Gilroy, CA) were used in the LRR studies. Mice were maintained under a 12-hr light/dark cycle with food and water ad libitum. In a given experiment, mice were randomly assigned to each test group. Testing was performed between 9:00 a.m. and 4:00 p.m. 3,5-P was tested at doses of 0, 1.25, 2.5, 5 and 7.5 mg/kg i.v. (injection volume is 30 µl) in 20% hydroxypropyl--cyclodextrin (Research Biochemicals, Natick, MA). At 30 sec after injection, each mouse was placed on its back. The observation period continued for 2 min after i.v injection. Any mice failing to right itself by returning to an upright position within 2 min was scored as having lost its righting reflex. Mice in the 5-THDOC + 3,5-P group were administered 5-THDOC (1 mg/kg i.v.) immediately before the injection of 3,5-P at 1.25, 2.5, 5, 7.5 or 12.5 mg/kg i.v., respectively. Mice receiving 5-THDOC alone did not show LRR at any time up to 30 min after the injection. The ED₅₀ values were determined according to the method of Litchfield and Wilcoxon (1949), and the significance of the potency ratio in the presence or absence of 5-THDOC was determined by the ² test.

	Results

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Effect of 5-THDOC on 3,5-P and 5-THDOC on the modulation of [³⁵S]TBPS binding to rat cortex and alpha-1-beta-2-gamma-2L subunit containing GRCs. In the presence of 5 µM GABA, 3,5-P, 5-THDOC and 5-THDOC inhibited [³⁵S]TBPS binding in rat cortex with IC₅₀ values of 29, 99 and 145 nM respectively (fig. 2). Consistent with previous results (Gee and Lan, 1991), the endogenous neuroactive steroids 3,5-P and 5-THDOC inhibit [³⁵S]TBPS binding in rat cortex with apparent full efficacy (i.e., 100% inhibition). As shown as in figure 2, 5-THDOC inhibited [³⁵S]TBPS binding in rat cortex with certain limited efficacy. In light of the difference in efficacy between 5-THDOC and the apparent full-efficacy neuroactive steroids 3,5-P and 5-THDOC, it was of interest to study the interactions among 5-THDOC, 3,5-P and 5-THDOC in the modulation of [³⁵S]TBPS binding. Fixed concentrations of 5-THDOC of 3 µM caused a parallel rightward shift in the dose-response curves for both 3,5-P and 5-THDOC displacement of [³⁵S]TBPS binding (fig. 3, A and B). The magnitude of the rightward shift for both curves was dependent on the concentration of 5-THDOC. When the data from these dose-response curves were subjected to Schild analysis (Schild, 1949), the pA₂ values were 6.7 for 3,5-P +5-THDOC and 7.04 for 5-THDOC + 5-THDOC (fig. 4). The slopes of the Schild plots were 0.99 for 3,5-P +5-THDOC and 0.98 for 5-THDOC +5-THDOC (fig. 4). On the basis of the pA₂ values, the apparent potencies of 5-THDOC as an antagonist of 3,5-P and 5-THDOC were ~200 and 90 nM, respectively. These values are in reasonable agreement with 5-THDOC IC₅₀ values of 145 nM against [³⁵S]TBPS binding when measured directly under similar assay conditions. The results are consistent with the hypothesis that 5-THDOC is a partial agonist at the neurosteroid site recognized by 3,5-P and 5-THDOC.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent inhibition of 2 nM [35S]TBPS binding to cortical P2 homogenates by 3,5-P , 5-THDOC  and 5-THDOC  in the presence of 5 µM GABA. Each point represents the mean ± S.E.M. of at least four independent experiments.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effect of various concentrations of 5-THDOC on 3,5-P and 5-THDOC inhibition of 2 nM [35S]TBPS binding to rat cortical P2 homogenates. A, 3,5-P modulation of [35S]TBPS binding in the presence of 0 (, IC50 = 29.7 nM), 0.3 (, IC50 = 83 nM), 0.6 (, IC50 = 152 nM), 1 (, IC50 = 207 nM) and 3 (, IC50 = 896 nM) µM 5-THDOC. The IC50 values are significantly different (ANOVA, P  .01). B, 5-THDOC modulation of [35S]TBPS binding in the presence of 0 (, IC50 = 99 nM), 0.1 (, IC50 = 174 nM), 0.3 (, IC50 = 319 nM), 1 (, IC50 = 872 nM) and 3 (, IC50 = 2315 nM) µM 5-THDOC. Each point represents the mean ± S.E.M. of four or five independent experiments. The IC50 values are significantly different (ANOVA, P  .05).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Schild regression plots for the antagonism of 3,5-P  and 5-THDOC  modulation of [35S]TBPS binding by 5-THDOC in rat cortex. DR is the ratio of the IC50 values for 3,5-P or 5-THDOC inhibition of [35S]TBPS binding in the presence or absence of 5-THDOC. The slopes are 0.99 and 0.98 for the 3,5-P and 5-THDOC plots, respectively. Data points represent the mean of four experiments.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effect of 1 (, IC50 = 208 nM) and 3 (, IC50 = 636 nM) µM 5-THDOC on 3,5-P (, IC50 = 319 nM) modulation of [35S]TBPS binding to recombinantly expressed alpha-1-beta-2- gamma-2L subunit-containing GRCs. Each point represents the mean ± S.E.M. of at least three independent experiments. The IC50 values are significantly different (ANOVA, P  .05).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Functional modulation of GABA-activated currents by 5-THDOC and 3,5-P in X. laevis oocytes expressing the GABAA receptor subunit combination alpha-1-beta-2- gamma-2L. Concentration-effect curves demonstrate low-efficacy potentiation of 5% GABA responses by 5-THDOC  relative to full-efficacy modulation by 3,5-P (). At concentrations of >0.3 µM, steroids activated currents (direct current) in the absence of GABA. These direct currents were larger for 3,5-P () than for 5-THDOC (). In all cases, n = 4.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Electrophysiological evidence that 5-THDOC blocks modulation induced by the full agonist 3,5-P. Left, sample records demonstrating surmountable inhibition of 3,5-P-induced modulation by 5-THDOC at alpha-1-beta-2- gamma-2L receptors in oocytes. The control response was ~5% of the maximal GABA current (not shown). Right, modulation by 1 µM 3,5-P (dashed line) was used to scale other currents in the analysis data; data from 10 experiments (see left) were pooled to illustrate blockade of 3,5-P-induced modulation by 5-THDOC. Far left column, slightly larger potentiation induced by 10 µM 3,5-P. Two right columns, coapplication of 3 µM 5-THDOC and 3,5-P at the concentrations noted. Values were for 3 µM 5-THDOC alone, 54.9 ± 2.4%; 3 µM 5-THDOC coapplied with 1 µM 3,5-P, 74.5 ± 2.6%; and 3 µM 5-THDOC coapplied with 10 µM 3,5-P, 91.9 ± 1.5% (n = 10 for all conditions and significantly different P  .01 by ANOVA).

Behavioral studies. Both 3,5-P and 5-THDOC has been reported to have central nervous system depressant actions, including sedative-hypnotic and anesthetic effects mediated through modulation of the GRC (Gee et al., 1995). Based on the in vitro studies, 5-THDOC could potentially have antagonist actions against full-efficacy neuroactive steroid such as 3,5-P in vivo. Consequently, the LRR in mice was used as a behavioral measure of GRC-mediated central nervous system depression. 3,5-P induced dose-dependent LRR with an ED₅₀ value of 3.5 mg/kg (table 1). 5-THDOC at 1 mg/kg (a dose that did not produce LRR alone) increased the ED₅₀ value for 3,5-P induced LRR to 7 mg/kg. The dose at which 5-THDOC induced the LRR was >2 mg/kg i.v. The solubility of 5-THDOC above this dose was limited, which prevented further evaluation of its ability to induce an LRR. These behavioral findings are consistent with 5-THDOC having antagonist action in vivo at the same site as 3,5-P on the GRC.

View this table: [in this window] [in a new window]   TABLE 1 The effect of 5-THDOC (1 mg/kg) on 3,5-P induced LRR in mice

	Discussion

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Previous studies have shown that 5-THDOC has limited efficacy in the modulation of [³⁵S]TBPS binding and ³⁶Cl uptake in the rat cortex and no efficacy in the spinal cord (Gee and Lan, 1991). 5-THDOC was also shown to antagonize 3,5-P modulation of [³⁵S]TBPS binding in rat spinal cord. Based on these data, it was proposed that 5-THDOC was either a partial agonist or a receptor subtype-selective ligand. In the present study, the basis for the apparent limited efficacy of 5-THDOC at the GRC was investigated in detail both in vitro and in vivo.

In binding assays, 5-THDOC behaves in a manner consistent with interaction with a site recognized by 5-THDOC and 3,5-P. The pA₂ values derived from Schild analysis are in the same range as the IC₅₀ values for 5-THDOC inhibition of [³⁵S]TBPS binding. These data suggest that 3,5-P, 5-THDOC and 5-THDOC act at the same site in rat cortex. However, interpretation of the data rat cortex is complicated by receptor heterogeneity (Laurie et al., 1992; Wisden et al., 1992). GABA_A receptor subtypes have different pharmacological properties (Draguhn et al., 1990; Smart, et al., 1991). In particular, subunit composition of the GRC produces apparent changes in the allosteric modulatory effects of neuroactive steroids (Lan et al., 1990, 1991; Puia et al., 1991; Shingai et al., 1991). Expression of GABA_A receptor subunits of known composition generates a homogeneous system for studying the interactions between 5-THDOC and 3,5-P. Using a system with defined subunit composition rules out the possibility that the apparent limited efficacy of 5-THDOC in cortical homogenates results from the modulation of a subpopulation of [³⁵S]TBPS binding sites. Consistent with the results observed in the cortex, 5-THDOC (1 and 3 µM) causes a rightward parallel shift of the 3,5-P/[³⁵S]TBPS dose-response curves without changing the degree of maximum inhibition. The magnitude of the shift in the IC₅₀ values for 3,5-P-induced by 5-THDOC is close to that observed in cortical homogenates. Combined, the data from cortical homogenates and expressed GRCs of known subunit composition provide strong support that 5-THDOC is a partial agonist acting at the same site as 3,5-P.

Electrophysiological studies similarly suggest that 5-THDOC has partial agonist activity in the modulation of GABA currents mediated by alpha-1-beta-gamma-beta-2-containing GRCs expressed in oocytes. Coapplication of 5-THDOC with 3,5-P results in significantly less potentiation of GABA currents than is observed with 3,5-P alone. Most importantly, this inhibition could be surmounted by increasing the concentration of the full agonist. In combination with the binding data, these observations strongly suggest that the antagonist activity of 5-THDOC is mediated by the neurosteroid binding site at which 3,5-P evokes full-efficacy modulation.

In vivo, 5-THDOC (1 mg/kg) induces a 2-fold increase in the ED₅₀ value for 3,5-P induction of LRR in mice. This dose of 5-THDOC alone did not produce LRR. Thus, the antagonist actions of 5-THDOC can be observed both in vitro and in vivo. However, the potency of 5-THDOC in blocking 3,5-P induction of LRR was observed to be greater than that in vitro. The possible explanations for the greater potency in vivo are additional factors such as different level of endogenous GABA or enhanced bioavailability of 5-THDOC.

Earlier studies have shown that certain non-3-hydroxylated neuroactive steroids interact with the GRC as allosteric antagonists of GABA action (Demirgoren et al., 1991; Majewska et al., 1986, 1988). These neuroactive steroids do not appear to produce their actions at the same site as 3,5-P (Gee et al., 1989). The existence of competitive neuroactive steroid antagonists with high affinity has not yet been unequivocally demonstrated. Although evidence has been presented to suggest that 5-pregnan-3-ol-20-one competitively antagonizes the action of 5-pregnan-3-ol-20-one as a modulator of [³H]flunitrazepam binding to the benzodiazepine receptor (Prince and Simmonds, 1992), the high concentration of steroid required to block the potentiation of benzodiazepine receptor binding, ~60 µM in these studies, suggests that the ideal structure-activity requirements for competitive antagonists have not yet been identified. The structural leads provided by 5-pregnan-3-ol-20-one and 5-THDOC may be useful avenues of approach in the search for a pure antagonist.

Collectively, the results of the present study strongly support the hypothesis that 5-THDOC is a partial agonist at the same site as the full agonists 5-THDOC and 3,5-P on the GRC. Both 3,5-P and 5-THDOC are detected in the rat brain (Purdy et al., 1991). Brain levels of these steroids after swim stress reach concentrations that are sufficient to potentiate GABA action when based on the in vitro levels necessary to modulate GABA action at the GRC (Gee et al., 1987; Majewska et al., 1986; Purdy et al., 1991). Whether sufficient levels of the 5-isomer are produced in the central nervous system to affect GRC function is uncertain, although 5-reductase activity has been detected in the central nervous system and periphery (Mickan, 1972; Mickan and Zander, 1979). It is possible, therefore, that 5-THDOC has a role in the endogenous modulation of the GABA_A receptor. The relative contribution of regional selectivity of 5-THDOC and 5-THDOC to the pharmacological profile is not currently known despite evidence suggesting that 5-THDOC shows regional selectivity (Gee and Lan, 1991). Nevertheless, it is possible that the pharmacological profile of 5-THDOC is a reflection of both its limited efficacy and its regional selectivity.

Especially intriguing is the question of the physiological role of a endogenous partial agonist ligand in the modulation of the GRC relative to those of full agonists. Moreover, whether apparent partial agonist neuroactive steroids of this type have unique pharmacological profiles relative to their full efficacy counterparts remains to be determined. Recently, the synthetic partial agonist neurosteroid 3-hydroxy-3-trifluoromethyl-5-pregnan-20-one was synthesized and characterized in vitro (Hawkinson et al., 1996). This synthetic neuroactive steroids may be a useful tool for the evaluation of the in vivo pharmacological profile of limited efficacy neuroactive steroids because of its lower efficacy and metabolic lability (i.e., rapid metabolic degradation and elimination) relative to 5-THDOC.

In conclusion, the present study is the first demonstration of competitive antagonism of endogenous neuroactive steroids acting on the GRC. Our findings that 5-THDOC can antagonize both in vitro and in vivo actions of neuroactive steroids on the GRC raise the possibility that appropriate modification of the 5-THDOC molecule may give rise to high-affinity steroids that are pure antagonists at the neuroactive steroid site on the GRC. Such antagonists will provide essential tools for the elucidation of the physiological role of endogenous neuroactive steroids.