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Vol. 60, Issue 4, 732-741, October 2001


Effects on gamma -Aminobutyric Acid (GABA)A Receptors of a Neuroactive Steroid That Negatively Modulates Glutamate Neurotransmission and Augments GABA Neurotransmission

Steven Mennerick, Chun-Min Zeng, Ann Benz, Weixing Shen, Yukitoshi Izumi, Alex S. Evers, Douglas F. Covey, and Charles F. Zorumski

Departments of Psychiatry (S.M., A.B., W.S., Y.I., C.F.Z.), Anatomy and Neurobiology (S.M., C.F.Z.), Molecular Biology and Pharmacology (C.-M.Z., A.S.E., D.F.C.), and Anesthesiology (A.S.E.), Washington University School of Medicine, St. Louis, Missouri

    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Neurosteroids positively and negatively modulate gamma -aminobutyric acid (GABA)A receptors and glutamate receptors, which underlie most fast inhibition and excitation in the central nervous system. We report the identification of a neuroactive steroid, (3alpha ,5beta )-20-oxo-pregnane-3-carboxylic acid (3alpha 5beta PC), with unique cellular actions. 3alpha 5beta PC positively modulates GABAA receptor function and negatively modulates N-methyl-D-aspartate (NMDA) receptor function, a combination that may be of particular clinical benefit. 3alpha 5beta PC promotes net GABAA potentiation at low steroid concentrations (<10 µM) and at negative membrane potentials. At higher concentrations, the steroid also blocks GABA receptors. Because this block would presumably counteract the NMDA receptor blocking actions of 3alpha 5beta PC, we characterize the GABA receptor block in some detail. Agonist concentration, depolarization, and high extracellular pH increase the block. The apparent pK for both potentiation and block was 6.4 to 6.9, substantially higher than expected from carboxylated steroid in an aqueous environment. Block is not dependent on the stereochemistry of the carboxylic acid at carbon 3 and is relatively insensitive to placement of the carboxylic acid at the opposite end of the steroid (carbon 24). Potentiation is critically dependent on the stereochemistry of the carboxylic acid group at carbon 3. Consistent with the pH dependence of potentiation, effects of the amide derivative (3alpha ,5beta )-20-oxo-pregnane-3-carboxamide, suggest that the un-ionized form of 3alpha 5beta PC is important for potentiation, whereas the ionized form is probably responsible for block. Further refinement of the neuroactive steroid to promote GABA potentiation and NMDA receptor block and diminish GABA receptor block may lead to a clinically useful neuroactive steroid.

    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Neurosteroids have received recent wide attention because of their endogenous presence in the central nervous system at concentrations that may modulate GABAergic and/or glutamatergic synaptic communication (Baulieu, 1998; Concas et al., 1998). Synthetic analogs of endogenous neurosteroids may be clinically useful neuroprotectants, anesthetics, and anticonvulsants (Gasior et al., 1999; Zorumski et al., 2000). Generally, augmentation of GABAergic transmission and block of NMDA receptor-mediated transmission are associated with anesthetic, anticonvulsant, and antiexcitotoxic properties (Macdonald and Greenfield, 1997). Unfortunately, whereas many neuroactive steroids have activity at both of these receptor types, no known endogenous or synthetic steroid dampens NMDA receptor signaling without also inhibiting GABAergic signaling. These are opposing cellular effects with regard to the clinically desirable properties mentioned above.

Pregnane steroid derivatives with a sulfate or other negatively charged substituent at the carbon 3 (C3) position in the alpha -configuration are negative modulators of NMDA receptors through noncompetitive, voltage-independent block (Park-Chung et al., 1994). The beta -configuration of the ring fusion at C5 is also important for blocking action, as beta  sulfate substitution at C3 retains NMDA receptor blocking activity if the steroid is 5beta -reduced (Park-Chung et al., 1994; Weaver et al., 2000). Hemisuccinate and other hemiester alpha -substituents at C3 retain NMDA receptor blocking activity (Weaver et al., 1997, 2000). Unfortunately, the neuroprotective profile of each of these NMDA receptor antagonists is compromised by the fact that each of these derivatives also blocks GABAA receptor activity (Park-Chung et al., 1999). Additionally, whereas (3alpha ,5beta )-3-hydroxypregnan-20-one hemisuccinate, the hemisuccinate analog of naturally occurring pregnanolone sulfate, has been shown to be neuroprotective (Weaver et al., 1997), this analog is subject to hydrolysis of the hemisuccinate group.

We report here the cellular characterization of a novel neurosteroid analog, (3alpha ,5beta )-20-oxo-pregnane-3-carboxylic acid (3alpha 5beta PC), with both NMDA antagonist and GABA-potentiating actions. We synthesized this steroid as a nonhydrolyzable analog of (3alpha ,5beta )-3-hydroxypregnan-20-one hemisuccinate (Weaver et al., 1997). Like the hemisuccinate, at physiological pH the carboxylic acid group of 3alpha 5beta PC should be largely deprotonated, making 3alpha 5beta PC a likely NMDA receptor blocker. However, like GABA-potentiating steroids, which contain a 3alpha -hydroxyl group as a hydrogen bond donor (Phillipps, 1975), the ---COOH of un-ionized 3alpha 5beta PC is a similarly located hydrogen bond donating group, and therefore un-ionized 3alpha 5beta PC might be expected to potentiate GABA receptors.

3alpha 5beta PC exhibits complex actions on GABAA receptors, with potentiation, voltage-dependent block, and direct gating of GABAA receptors. At low concentrations of drug and at physiological pH, net potentiation of GABAA receptor function and IPSCs are observed. At higher concentrations, the postsynaptic GABAA potentiation is decreased by steroid-induced block of receptor function. Block shows little stereoselectivity and is relatively insensitive to placement of the carboxylate at either end of the steroid ring structure. The pH dependence of both block and potentiation by carboxylated steroids suggests a higher apparent pK than expected of these organic acids in water. This probably reflects the influence of membrane/protein constituents on the pK of the carboxylate group.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Hippocampal Cultures. Primary hippocampal microcultures were prepared from 1- to 3-day-old postnatal albino rats using established methods (Mennerick et al., 1995). Under halothane anesthesia, rats were decapitated, and the hippocampi were dissected and cut into 500-µm-thick transverse slices. The slices were dissociated with 1 mg/ml papain in oxygenated Leibovitz L-15 medium and mechanically triturated in modified Eagle's medium containing 5% horse serum, 5% fetal calf serum, 17 mM D-glucose, 400 µM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. Isolated cells were plated (75 cells/mm2) onto plastic culture dishes coated first with 0.15% agarose then with atomized droplets of rat tail collagen. To halt glial proliferation, cultures were treated with 10 µM cytosine arabinoside after 3 days in vitro. Experiments were performed in cultures that were 4 to 14 days old.

Xenopus laevis Oocytes. Stage V-VI oocytes were harvested from sexually mature female X. laevis (Xenopus One, Northland, MI) under 0.1% tricaine (3-aminobenzoic acid ethyl ester) anesthesia. Oocytes were defolliculated by shaking for 20 min at 37°C in collagenase (2 mg/ml) dissolved in calcium-free solution containing: 96 mM NaCl, 2 mM KCl, 1 mM MgCl2 and 5 mM HEPES at pH 7.4. Capped mRNA, transcribed in vitro (mMessage mMachine; Ambion, Austin, TX) from linearized plasmids containing receptor-coding regions, were injected into oocytes 24 h after defolliculation. Oocytes were incubated for up to 2 weeks at 18°C in ND96 medium containing 96 mM NaCl, 1 mM KCl, 1 mM MgCl2, 2 mM CaCl2, and 10 mM HEPES at pH 7.4 supplemented with 5 mM pyruvate and the above-mentioned antibiotics. The cDNAs for the GABAA receptor subunits were provided by A. Tobin [University of California, Los Angeles (alpha 1)], P. Malherbe [Hoffman-La Roche, Switzerland (beta 2)], and C. Fraser [National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD (gamma 2L)].

Electrophysiology. For synaptic studies, the growth medium was exchanged for a solution containing 138 mM NaCl, 4 mM KCl, 10 mM HEPES, 10 mM D-glucose, 2 mM CaCl2, and 1 mM MgCl2, pH 7.25. Whole-cell voltage-clamp recordings of autaptic currents were performed from neurons on single-neuron islands using recording pipettes with open tip resistances of 2 to 5 MOmega . The pipette solution contained 140 mM KCl, 4 mM NaCl, 5 mM EGTA, 0.5 mM CaCl2, 10 mM HEPES, pH 7.25. Neurons were recorded using an Axopatch 1-D patch-clamp amplifier (Axon Instruments, Foster City, CA), and series resistance (<10 MOmega ) was compensated 90 to 100% during experiments. Synaptic transmission was activated by stimulating neurons with 1.5-ms voltage steps from -70 to +30 mV at intervals of 20 to 30 s. During experiments, microislands were continuously superfused locally using a gravity-driven multibarrel pipette with a common exit port. The tip of this local perfusion system was placed ~400 µm from the microisland being recorded, and solution flowed at a rate of 0.1 to 0.5 ml/min. All recordings were done at room temperature (~22°C) on the stage of a Nikon inverted microscope equipped with phase-contrast optics. Studies examining exogenous applications of agonists were performed using whole-cell recordings as described above, except the pipette solution contained 140 mM CsCl in place of KCl. Experiments examining low-Mg2+-induced action potentials (Fig. 1F) were performed in the current-clamp mode of the patch amplifier on islands containing small networks of neurons.


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Fig. 1.   Synaptic effects of 3alpha 5beta PC. A, structure of 3alpha 5beta PC showing the pregnane core and stereochemistry of the C3 and C5 substituents. B and C, 3alpha 5beta PC selectively depressed the slow NMDA receptor component of EPSCs. B, autaptic EPSCs were elicited from solitary glutamatergic neurons in microcultures with a 2-ms voltage pulse to 0 mV (from a holding potential of -70 mV). The extracellular bath solution contained no added Mg2+ and 10 µM glycine to unmask NMDA receptors. The traces represent average responses obtained in the absence (thin trace) and presence (thick trace) of 50 µM 3alpha 5beta PC. Note that the peak EPSC was not affected by the drug, but the slow NMDA component was selectively diminished. Transient currents associated with stimulation have been blanked in this and subsequent figures for clarity. C, summary of the effects of 50 µM 3alpha 5beta PC on peak EPSC and the NMDA component (measured 30 ms after stimulation) in nine neurons. D and E, concentration-response effects of 3alpha 5beta PC on hippocampal IPSCs. D, top to bottom, represent the effect of 3alpha 5beta PC on an IPSC from a solitary GABAergic cell (all from the same cell). In all cases, the thick trace represents the IPSC in the presence of drug, whereas the thin trace represents the IPSC in the absence of drug. As noted in the text, the effect on peak amplitude seen with 50 µM 3alpha 5beta PC was variable from cell to cell. E, summary data representing the concentration dependence of 3alpha 5beta PC on IPSC 10 to 90% decay time. Asterisks denote significantly different than zero (p < 0.05, two-tailed t test). The values for the 10 to 90% decay times from which the normalized data in Fig. 1E were derived were baseline, 114 ± 13 ms; 1 µM 3alpha 5beta PC, 105 ± 10 ms; 5 µM 3alpha 5beta PC, 143 ± 10 ms; 10 µM 3alpha 5beta PC, 132 ± 7 ms; and 50 µM 3alpha 5beta PC, 101 ± 16 ms. F, in a current-clamp recording from a small network of neurons grown in a microculture, action potential activity was increased by removing extracellular Mg2+ and adding glycine to potentiate NMDA receptor activity (low Mg2+). 3alpha 5beta PC (5 µM) reversibly eliminated the firing. The apparent increase in firing upon removal of drug in this cell was not consistently observed in other neurons.

For oocyte recordings, experiments were performed with a virtual ground, two-electrode voltage clamp using a Dagan CA-1B amplifier 1 to 10 days after RNA injection. The extracellular recording solution was ND96 medium (with no supplements). Intracellular recording pipettes were filled with 3 M KCl and had open tip resistances of ~1MOmega . Drugs were applied from a common tip via a gravity-driven multibarrel drug-delivery system. Acetic acid and NaOH were used to adjust the pH of extracellular solutions.

For synaptic studies, averages of two to eight traces per experimental condition were used for analysis and display. Currents were filtered at 1 to 5 kHz using a four-pole Bessel filter and were digitized using pClamp, version 6.0 (Axon Instruments). Data were analyzed off-line using the pClamp software. Unless otherwise noted, results represent mean ± S.E.M. Statistical differences were determined using two-tailed t tests.

Drugs. Unless otherwise stated, drugs were from Sigma (St. Louis, MO). Pregnenolone sulfate and (3alpha ,5beta )-pregnan-20-one sulfate (3alpha 5beta PS) were obtained from Steraloids (Newport, RI) and from Sigma. The 3beta 5beta PS was prepared as described elsewhere (Park-Chung et al., 1997) and was the generous gift of Dr. Robert H. Purdy (Scripps Research Institute, La Jolla, CA). A preliminary account of the synthesis of 3alpha 5beta PC and 3beta 5beta PC has been published (Zeng et al., 1999). Full synthetic details will be published elsewhere. The methyl ester of 3alpha 5beta PC was prepared by reacting the acid with diazomethane dissolved in ether. The (3alpha ,5beta )-20-oxo-pregnane-3-carboxamide (3alpha 5beta PA) was prepared from 3alpha 5beta PC by converting this carboxylic acid to the acid chloride and then reacting this intermediate with ammonia dissolved in methylene chloride.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Alteration of Synaptic Activity by 3alpha 5beta PC. Figure 1A shows the structure of 3alpha 5beta PC. 3alpha 5beta PC selectively depressed the slow NMDA receptor component of EPSCs in hippocampal neurons, consistent with the effect of other neuroactive steroids with a charged moiety in the alpha -configuration at C3 (Park-Chung et al., 1997). 3alpha 5beta PC (20 µM) produced only 8 ± 5% depression of the NMDA component (N = 3; data not shown), whereas 50 µM 3alpha 5beta PC produced 20 ± 4% depression (N = 9; Fig. 1, B and C). Effects on the fast alpha -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor component of EPSCs were negligible with 8 ± 5% potentiation produced at 50 µM (Fig. 1, B and C).

To explore the effects of 3alpha 5beta PC on GABA transmission, we examined the effect of 3alpha 5beta PC on GABAergic IPSCs in solitary hippocampal neurons. Figure 1D shows the effect of 3alpha 5beta PC on GABAergic IPSCs over a range of concentrations. Clear potentiating effects were observed at moderately low concentrations, making 3alpha 5beta PC the first described neuroactive steroid to both block NMDA receptor-mediated neurotransmission and potentiate GABAergic neurotransmission. In 10 of 14 neurons examined with 50 µM 3alpha 5beta PC, the peak amplitude of the IPSC was reversibly increased by >25% (106 ± 52% increase; N = 14). However, because of variability in the magnitude of this effect, changes in the peak amplitude reached only trend-level significance (p = 0.07; N = 14). The more prominent effect of the steroid was the prolongation of IPSC decay time course (Fig. 1D), similar to actions of other neuroactive steroids and other GABAA potentiators. However, the effects of 3alpha 5beta PC on IPSC decays decreased at higher concentrations, yielding a bell-shaped concentration-response relationship (Fig. 1E). At concentrations >10 µM, the prolongation of IPSCs by 3alpha 5beta PC became less apparent, such that effects of 50 µM 3alpha 5beta PC were not significantly different than in the absence of drug (Fig. 1E).

In all neurons tested, regardless of whether they exhibited a glutamatergic or GABAergic autaptic postsynaptic current, higher concentrations of steroid gated a steady inward current probably resulting from direct gating of GABAA receptors by 3alpha 5beta PC, a common effect of GABA-potentiating neuroactive steroids (Majewska, 1992). In five GABAergic cells, the respective concentrations of 5, 10, and 50 µM 3alpha 5beta PC gated currents of 15 ± 8, 33 ± 14, and 56 ± 26 pA. Although we did not study the directly gated current in detail, it is possible that this tonic activation of GABAA receptors could be physiologically and clinically significant (Bai et al., 2001).

To verify that the net cellular effect of 3alpha 5beta PC is indeed inhibitory, we examined the effect of 3alpha 5beta PC on action potential firing activity in small groups of hippocampal neurons grown in microcultures. Firing within networks was induced by lowering extracellular Mg2+ to 0.1 mM and adding saturating concentrations of extracellular glycine to augment NMDA receptor function. Similar conditions have been previously shown to enhance action potential activity in primary cultures (Segal and Furshpan, 1990). Concentrations between 5 and 50 µM 3alpha 5beta PC stopped low Mg2+-induced spiking in all cells tested (N = 7). The effect of 5 µM 3alpha 5beta PC on one cell is depicted in Fig. 1F.

3alpha 5beta PC Potentiates and Blocks GABAA Receptors. In subsequent experiments we sought to explain the complicated actions of 3alpha 5beta PC on IPSC decays using a combination of biophysical and pharmacological approaches. Prolongation of the decay phase of IPSCs is a common effect of GABA potentiators, including neuroactive steroids, benzodiazepines, and barbiturates, and probably underlies at least part of the clinical utility of these drugs. The reversal of IPSC prolongation at high steroid concentrations thus presumably undermines a potentially important property of the drug, especially because these high concentrations of steroid overlap with the concentrations that affect NMDA receptor-mediated EPSCs. We therefore probed structural and functional mechanisms of IPSC block by 3alpha 5beta PC in more detail.

We hypothesized that 3alpha 5beta PC possesses both potentiating and blocking activity at postsynaptic GABAA receptors, with potentiating effects dominating at low concentrations of drug and block dominating at higher concentrations. This would explain the apparent reversal of IPSC prolongations at higher concentrations (Fig. 1, D and E). To verify that postsynaptic effects explain the effects observed on both EPSCs and IPSCs, we used exogenous applications of agonists at AMPA/kainic acid, NMDA, and GABA receptors to isolate postsynaptic effects of 3alpha 5beta PC (Fig. 2). Consistent with synaptic data, at -70 mV there was no effect of 3alpha 5beta PC on responses to 100 µM kainic acid, a nondesensitizing AMPA receptor agonist. Again, consistent with synaptic data, block of NMDA responses (Fig. 2B) and potentiation of GABA receptor responses (Fig. 2C) were prominent at negative membrane potentials. Interestingly, upon washout of drug during GABA applications, a small off-response was noted in all cells tested (Fig. 2C; N = 5). This could reflect the hypothesized blocking action of 3alpha 5beta PC, which upon washout is relieved faster than potentiation is relieved.


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Fig. 2.   Postsynaptic effects of 3alpha 5beta PC reveals block of NMDA receptors and both potentiation and block of GABAA receptors. A, responses to the nondesensitizing AMPA receptor agonist kainic acid (KA, 100 µM) at two membrane potentials are represented. There was no effect of 3alpha 5beta PC (50 µM) at either potential. B, responses to 100 µM NMDA in another neuron. Extracellular Mg2+ was omitted and 10 µM glycine was added to all solutions. In addition, extracellular calcium concentration was lowered to 0.2 mM to reduce Ca2+-dependent desensitization of NMDA receptors (Clark et al., 1990; Legendre et al., 1993). 3alpha 5beta PC (50 µM) inhibited responses to NMDA with little voltage dependence. C, responses to GABA (2 µM) in the absence and presence of 50 µM 3alpha 5beta PC. Note the potentiation of GABA responses at -70 mV but block of responsiveness at + 40 mV. D, responses to 1 mM GABA at -70 mV in the absence (left) and presence (right) of 10 µM 3alpha 5beta PC.

In an attempt to isolate the GABAA receptor blocking effect, we reasoned that the ionized (---COO-) form of 3alpha 5beta PC should be prominent at physiological pH and that the associated negative charge may impart a voltage dependence to any GABA receptor-blocking effects of the drug. Consistent with a voltage-dependent block of GABAA receptors by 3alpha 5beta PC, we observed that at positive membrane potentials, instead of potentiation, net inhibition of GABA responses was observed (Fig. 2C). No overshooting off-response was observed at the positive potential, possibly reflecting slower relief from block at the positive potential (Fig. 2C). There was no apparent voltage dependence to the blocking effect of 50 µM 3alpha 5beta PC at NMDA receptors (Fig. 2B; 42 ± 3% depression at -70 mV, N = 6 and 45 ± 3% depression at +40 mV, N = 4; p > 0.5).

If 3alpha 5beta PC-mediated block of GABA receptors explains the reversal of IPSC prolongations at concentrations >5 µM then the 3alpha 5beta PC may block GABA receptors at negative membrane potentials or with application of high concentrations of GABA, such as are thought to be achieved briefly in the synaptic cleft during GABA neurotransmission. We found that when preapplied or coapplied with 1 mM GABA, 10 µM 3alpha 5beta PC produced no obvious potentiation at -70 mV (N = 7). Rather, 3alpha 5beta PC depressed peak GABA responses (by 24 ± 4%) and increased the rate of apparent desensitization (Fig. 2D). The 10 to 90% decay time during GABA applications was speeded by 58 ± 4% in the presence of steroid (N = 7). Taken together, these results suggest that block of 3alpha 5beta PC is fostered by high steroid concentrations, high GABA concentrations, and probably positive membrane potentials. Although several explanations for dependence of 3alpha 5beta PC block on GABA concentration are possible, further evidence for apparent use dependence and voltage dependence of block are presented below (Figs. 3 and 4)


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Fig. 3.   Potentiation is stereoselective about C3 but block is not. A, structure of 3alpha 5beta PC with emphasis (arrow) on the stereochemistry of the C3 substituent. B, leak subtracted GABA currents from a hippocampal neuron. A voltage pulse from -70 to +50 mV (top trace) was delivered in the absence and presence of 2 µM GABA. The current in the absence of GABA was digitally subtracted to reveal the GABA-gated current (bottom trace). Note the outward voltage-dependent relaxation of the GABA current. C, same protocol was applied to the same cell, except that 3alpha 5beta PC (50 µM) was also present in the GABA solution. Note the steady-state potentiation of GABA current apparent at -70 mV (before the voltage pulse) and the potentiation of the instantaneous GABA current observed at +50 mV, which gives rise to an inward relaxation and block of current relative to B at steady state. D-F, I/V relationships obtained from pulses to various membrane potentials. D, I/V relationship for GABA in the absence of modulator. Instantaneous current was measured between 3 and 5 ms after the voltage pulse. Steady-state current was measured at the end of the 200-ms voltage pulse. Note the nearly linear instantaneous I/V and outward rectification of the steady-state I/V. E, instantaneous I/V for GABA alone is replotted from D with the instantaneous current I/V relationship obtained in the presence of GABA plus 50 µM 3alpha 5beta PC. F, steady-state current I/V relationship for GABA alone is replotted from D along with the steady-state I/V relationship measured in the presence of GABA and 50 µM 3alpha 5beta PC. G-L, same protocols as in A-F were applied to a different cell using the diastereomer 50 µM 3beta 5beta PC. Note that potentiation is absent from both the instantaneous and steady-state currents at all potentials, but block is similar to that observed with 3alpha 5beta PC.

We were able to separate potentiation and block of GABAA receptors by examining current relaxations in response to voltage pulses in the presence of 2 µM GABA plus or minus 10 to 50 µM 3alpha 5beta PC. Voltage-dependent conductances in hippocampal neurons were inhibited with a combination of extracellular tetrodotoxin (500 nM) and Cd2+ (50 µM) to block sodium and calcium conductances, respectively, and intracellular Cs+ to block potassium conductances. Residual voltage-gated membrane conductances and leak currents were subtracted from GABA-induced currents digitally offline. GABA current/voltage (I/V) curves examined at membrane potentials between -50 and +50 mV showed outward rectification of the steady-state GABA current, similar to that seen with many GABA receptor subunit combinations (Segal and Barker, 1984; Burgard et al., 1996; Fig. 3, B and D) and a nearly linear instantaneous I/V relationship, measured immediately after the step to the test potential (Fig. 3D). The linear instantaneous I/V relationship suggests that the outward rectification observed in steady-state I/V curves derives from a voltage dependence of GABA binding or gating steps rather than from inherent rectification of single-channel conductance (Fig. 4). In the presence of 3alpha 5beta PC, potentiation dominated at negative membrane potentials in both the steady-state and instantaneous I/V relationships (Fig. 3, E and F). In contrast, at positive potentials, block of steady-state responses was apparent at positive membrane potentials (Fig. 3F). Inspection of raw traces revealed an inward, time-dependent relaxation of GABA currents to a steady-state level smaller than that gated by GABA alone (Fig. 3C). These results are consistent with a voltage-dependent (and/or gating-dependent) and time-dependent block of GABA receptors by 3alpha 5beta PC.


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Fig. 4.   Voltage and [GABA] dependence of 3beta 5beta PC block. A1, I/V relationship obtained from steady-state responses to 2 µM GABA at various membrane potentials from an X. laevis oocyte expressing recombinant GABAA receptors (alpha 1beta 2gamma 2L subunit combination). A voltage-pulse subtraction protocol similar to that used in hippocampal neurons (Fig. 3) was used. , GABA alone. open circle , currents in response to GABA plus 10 µM 3beta 5beta PC. A2, data in the presence of 3beta 5beta PC were replotted on an expanded y-axis to highlight the shape of the I/V curve. B1 and B2, same protocols applied to the same oocyte in A, except that 100 µM GABA was used as the agonist (plus 10 µM 3beta 5beta PC for open circles). Note the linearity of the 100 µM GABA I/V curve and the persistence of voltage-dependent block.

Structural Requirements for Block and Potentiation. Stereoselectivity of 3alpha 5beta PC effects on GABA receptors was examined to gain insight into the stereochemical requirements for potentiation and block. We synthesized the 3beta -diastereomer (3beta 5beta PC; Fig. 3G) and characterized 3beta 5beta PC in the same voltage-pulse protocol used to examine 3alpha 5beta PC. Interestingly, 3beta 5beta PC exhibited voltage-dependent block similar to that exhibited by the 3alpha -diastereomer, but the 3beta -compound elicited no potentiation (Fig. 3, H-L). These results suggest that potentiation is highly stereoselective whereas block is not.

With the benefit of a diastereomer that exhibited only block, we were able to examine the voltage dependence and [GABA] dependence of block in more detail. For these experiments we examined block of recombinant receptors expressed in X. laevis oocytes, which have the advantage of small background conductances and fewer concerns about spatial voltage clamp of neuronal dendritic trees. When examined in X. laevis oocytes expressing the alpha 1beta 2gamma 2L subunit combination, GABA I/V curves in response to 2 µM GABA were very similar to those obtained in hippocampal neurons. Figure 4A shows a steady-state I/V relationship to 2 µM GABA from a representative oocyte. The steady-state currents outwardly rectified, as observed in hippocampal neurons (Fig. 3). 3beta 5beta PC (10 µM) exhibited block at positive potentials but had almost no effect at negative potentials. Although this pattern of block could represent inherent voltage dependence to 3beta 5beta PC block, it is possible that the block at positive potentials is related directly to the increased gating of GABA receptors at positive potentials (evident in the outwardly rectifying I/V relationship). By this hypothesis, voltage dependence of block occurs indirectly because of the apparent use dependence of 3beta 5beta PC block.

To determine whether there is inherent voltage dependence of 3beta 5beta PC block, we examined the I/V relationship of responses to a high concentration of GABA (100 µM). The EC50 concentration for GABA in our experimental conditions was ~10 µM with a Hill coefficient near 2 (data not shown). Therefore, 100 µM GABA represents a concentration nearly maximum. Figure 4B shows I/V relationships for 100 µM GABA in the presence and absence of 10 µM 3beta 5beta PC obtained from the same oocyte represented in A. Note that the steady-state current in the absence of steroid increased ~34-fold at -90 mV (from 0.6 to 21.5 µA), consistent with a much higher probability of channel opening at 100 µM GABA. The I/V relationship for 100 µM GABA was nearly linear, in contrast to the outwardly rectifying I/V relationship for 2 µM GABA (Fig. 4, A1 and B1, solid symbols). This result suggests that the voltage-dependent steps in GABA receptor gating are no longer rate limiting in the presence of high concentrations of GABA. However, block by 3beta 5beta PC still exhibited notable voltage dependence. In contrast to the linear I/V curve for GABA alone, the I/V curve for 100 µM GABA in the presence of 10 µM 3beta 5beta PC exhibited inward rectification (Fig. 4B2). This result suggests that 3beta 5beta PC block possesses inherent voltage dependence, separate from its apparent use dependence. In addition, block by 10 µM 3beta 5beta PC at negative potentials, although negligible in the presence of 2 µM GABA, was dramatically increased in the presence of 100 µM GABA (Fig. 4, A1 and B1). Thus, 3beta 5beta PC block also exhibits clear dependence upon GABA concentration.

To further test the structural requirements for block by carboxylated steroids, we examined lithocholic acid, a bile steroid with a carboxylate group at C24. Lithocholic acid, like 3beta 5beta PC, blocked GABA receptors in a [GABA]-dependent manner (Fig. 5, B and C), suggesting GABA receptor block is relatively insensitive to placement of the carboxylate. Lithocholic acid was apparently a somewhat weaker blocker of GABAA receptors than 3beta 5beta PC, with 50 µM lithocholic acid inhibiting GABA responses by 71 ± 6% (N = 6) compared with 84 ± 3% block by 3beta 5beta PC under the same conditions (Fig. 5C; +90 mV, 20 µM GABA). Interestingly, we observed no evidence of potentiation of GABA responses by lithocholic acid at any voltage or GABA concentration. This result, similar to the results of C3 diastereomers (Fig. 3), suggests that potentiation is more susceptible to structural modifications of carboxylated steroids than block.


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Fig. 5.   Effect of lithocholic acid on GABA responses. A, structure of lithocholic acid with emphasis on placement of the carboxylate group (arrow) at C24. B, effect of lithocholic acid on responses to 20 µM GABA in an oocyte expressing the alpha 1beta 2gamma 2 subunit combination. C, summary of the GABA-dependence of lithocholic acid. Average block (10 µM lithocholic acid) of responses to 2 and 100 µM GABA is shown.

The results of Figs. 2 to 5 suggest that block of GABA receptors probably explains the complicated concentration effects of 3alpha 5beta PC on IPSCs (Fig. 1, D and E). As a direct test that GABA receptor block is relevant to IPSCs, we examined the effect of 3beta 5beta PC on IPSCs. The 3beta 5beta PC diastereomer (50 µM) truncated the time course of IPSCs as expected (Fig. 6, A and B). In five neurons, the peak IPSC was depressed by 3beta 5beta PC by 16 ± 3%. The 10 to 90% decay time was decreased by 49 ± 5%, from 112 ± 10 to 56 ± 3 ms. This result is consistent with the idea that the blocking action of 3alpha 5beta PC can explain the apparent reversal of IPSC prolongations at high steroid concentrations.


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Fig. 6.   3beta 5beta PC truncated the decay of IPSCs. A, thin trace represents control IPSCs. The thick trace represents averaged sweeps in the presence of 50 µM 3beta 5beta PC. B, summary of effects of 50 µM 3beta 5beta PC on the peak amplitude, 10 to 90% decay time, and total charge transfer from five neurons.

Dependence of Potentiation and Block on pH. Studies of the structural requirements of neuroactive steroid potentiation and block have previously suggested that a hydrogen bond donor at C3 is necessary for potentiation, whereas a negative charge at C3 is important for block (Phillipps, 1975). The predicted pK of the carboxylate group in 3alpha 5beta PC and 3beta 5beta PC is ~5.0 (Fini et al., 1987; Loudon, 1995). However, the pK of organic acids is dramatically altered by changing the solvent dielectric constant or hydrogen bonding ability (Fini et al., 1987; Smejtek et al., 1987). To explore the role of the ionization state of 3alpha 5beta PC in the potentiating and blocking action at GABAA receptors, we examined the effect of altered extracellular pH on 3alpha 5beta PC potentiation and block. For these studies, we examined the effect of steroids on X. laevis oocytes expressing the GABAA receptor subunit combination alpha 1beta 2gamma 2L, because these cells tolerate large and repeated shifts in pH (Fig. 7). As in hippocampal neurons, 50 µM 3alpha 5beta PC potentiated oocyte responses to 2 µM GABA at -70 mV and physiological pH (Fig. 7, A1 and A3). Consistent with previous results, low pH usually diminished responsiveness to GABA (Fig. 7A2; Zhai et al., 1998). Importantly, lowering the pH to 5.8 increased the net potentiation of 3alpha 5beta PC measured at -70 mV. Examination of steady-state I/V curves at pH 7.4 showed evidence for potentiation at negative membrane potentials and block at positive membrane potentials, again nearly identical to data from hippocampal neurons (Fig. 7A3). At pH 5.8, steady-state GABA responses were potentiated at all membrane potentials (Fig. 7A4). This result suggests that un-ionized 3alpha 5beta PC may relieve block, augment potentiation, or both.


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Fig. 7.   Potentiating effect of 3alpha 5beta PC was pH-dependent. A1 and A2, traces from an X. laevis oocyte expressing an alpha 1beta 2gamma 2L subunit combination. A1, at pH 7.4 and a membrane potential of -70 mV, 50 µM 3alpha 5beta PC potentiated the response to 2 µM GABA. A2, at the same membrane potential but pH 5.8, the response to GABA alone was slightly reduced but the potentiation is much larger than at the higher pH. A3 and A4, from the same cell, steady-state I/V curves showed loss of block and apparent enhanced potentiation at low pH. A3, steady-state I/V curve at pH 7.4. The potentiation and block at negative and positive membrane potentials at pH 7.4 are similar to results in hippocampal neurons (Fig. 3F). A4, steady-state I/V curve at pH 5.8. Note the potentiation of steady-state GABA responses at all membrane potentials. B, titration of the potentiating effect of 3alpha 5beta PC. Potentiation, independent of block, was isolated by using low concentrations of GABA (2 µM) and 3alpha 5beta PC (5 µM) and by examining negative membrane potentials -70 mV. Potentiation was calculated relative to the steady-state GABA response at each pH value. The solid line represents a titration curve with an apparent pK of 6.4. C1 and C2, potentiation of the amide derivative of 3alpha 5beta PC is pH-independent. Protocol was similar to that used for A1 and A2. The inset shows the structure of 3alpha 5beta PA. Holding potential was -70 mV.

To determine whether potentiation, independent of block, is affected by pH, we isolated the potentiating effect of 3alpha 5beta PC with low GABA concentrations (2 µM), low 3alpha 5beta PC concentration (5 µM), and a negative membrane potential (-70 mV). As shown by the titration curve in Fig. 7B, potentiation grew as pH was lowered, but the apparent pK was higher than predicted for an organic acid in water (pK of ~5). The apparent pK of 3alpha 5beta PC when interacting with receptor was ~6.4 from the fit to data in Fig. 7B. The saturation of responses at low pH values did not result from achieving maximum potentiation by steroid, because at pH 5.8, doubling the total steroid concentration from 5 to 10 µM increased the GABA responses a further 3.3 ± 0.2-fold (N = 3 oocytes; data not shown).

Potentiation by 3alpha 5beta PC does not seem dependent upon charge neutrality per se at C3, as the methyl ester derivative of 3alpha 5beta PC, which is electroneutral at C3 but is not a hydrogen bond donor, was inactive at concentrations up to 50 µM. In three oocytes treated with 2 µM GABA, responses at -50 mV were potentiated by only 8 ± 9% a 50 µM concentration of the methyl ester derivative, compared with 391 ± 10% potentiation for 50 µM 3alpha 5beta PC under the same conditions (N = 4; data not shown).

On the other hand, 3alpha 5beta PA, the amide derivative of 3alpha 5beta PC, which is electroneutral at C3 and a weaker hydrogen bond donor than un-ionized 3alpha 5beta PC, potentiated responses to 2 µM GABA in a concentration-dependent manner (Fig. 6C). In three X. laevis oocytes examined, responses were potentiated by 23 ± 7, 233 ± 47, and 326 ± 70% at 1, 10, and 50 µM, respectively. Unlike 3alpha 5beta PC, steady-state I/V plots of responses to 2 µM GABA in the presence of 50 µM 3alpha 5beta PA were potentiated at all potentials, with no evidence of block at positive potentials (data not shown). Also unlike 3alpha 5beta PC, potentiation by a subsaturating concentration of 3alpha 5beta PA showed very little pH dependence (cf. Figure 6, A and C). At pH 7.4, 5 µM 3alpha 5beta PA potentiated responses to 2 µM GABA by 75 ± 7%; at pH 5.8 responses were potentiated by 109 ± 14% (N = 3 oocytes). This result is consistent with the expectation that 3alpha 5beta PA is not ionized over the entire pH range examined. The result is also consistent with the hypothesis that the increased 3alpha 5beta PC potentiation at low pH results from a change in the concentration of un-ionized steroid and from titration of a residue on the receptor protein. Consequently, the high pK value for 3alpha 5beta PC suggests that the environment in which the bound steroid is located has a lower dielectric constant or hydrogen bonds more weakly than water (Smejtek et al., 1987).

To test the effect of low pH on GABA receptor block, independent of potentiation, we examined the effect of low pH on block by the 3beta -diastereomer and lithocholic acid (Fig. 8). We used a combination of 20 µM GABA and 50 µM 3beta 5beta PC to induce severe block across a range of membrane potentials. 3beta 5beta PC (50 µM) at -90 mV depressed responses to 2 µM GABA by 28 ± 7% but depressed responses to 20 µM GABA by 67 ± 6% (N = 5 oocytes at each GABA concentration). These data are consistent with the observed dependence of block by 3alpha 5beta PC on GABA concentration (Figs. 2, C and D, and 4). Surprisingly, despite the large block with elevated GABA concentration, low pH (5.8) caused nearly complete relief from block at all membrane potentials (Fig. 8, A and B). Depression of GABA responses was 84 ± 3% at +90 mV and pH 7.4, but depression was only 3 ± 3% at +90 mV and pH 5.8 (N = 5; Fig. 8D). Lithocholic acid behaved similarly to 3beta 5beta PC (Fig. 8C). As with 3alpha 5beta PC potentiation, titration curves for both 3beta 5beta PC and lithocholic acid (Fig. 8, B and C) revealed high apparent pK values (6.9 and 6.4, respectively).


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Fig. 8.   A1-A4, low pH diminished block by 3beta 5beta PC. Voltage pulses in the presence and absence of GABA were applied in 20-mV increments to potentials between -90 and +90 mV. The traces shown are subtractions of traces in the absence of GABA from those in the presence of GABA. A1, at pH 7.4 responses to 20 µM GABA showed typical outward rectification. Twenty µM GABA was used to enhance the effect of 3beta 5beta PC. A2, 3beta 5beta PC (50 µM) depressed GABA responsiveness at all membrane potentials. A3, GABA responsiveness was depressed slightly by pH 5.8. A4, 3beta 5beta PC depression was nearly eliminated at low pH. B and C, pH dependence of block by 3beta 5beta PC (B, N = 4) and by lithocholic acid (C, N = 5). Apparent pK values for the blockers were 6.9 and 6.4, respectively. D, low pH only slightly affected the block of GABA responses by 3alpha 5beta PS (2 µM). Because the block by 3alpha 5beta PS, like that of 3beta 5beta PC, is [GABA]-dependent, the small amount of reduction in effectiveness at low pH could be due to the decreased GABA responsiveness at pH 5.8. The summary reflects fractional block of steady-state response to 20 µM GABA at +90 mV for 50 µM 3beta 5beta PC (N = 5; different sample than Fig. 8B) and 2 µM 3alpha 5beta PS (N = 4).

To determine whether the ionization state of a receptor residue probably contributes to the effects of pH on 3beta 5beta PC block, we examined the effect of pH on several sulfated steroids whose blocking actions are similar to those of 3beta 5beta PC and probably act at a similar site. Ionization of these steroids, even if the local environment shifts the pK value by 2 units, should not be affected between pH 7.4 and 5.8 because of the extremely low pK of the sulfate group (Loudon, 1995). At pH 5.8, block of GABA (20 µM) responses by 2 µM 3alpha 5beta PS was decreased only slightly at all membrane potentials examined (Fig. 8D). Like 3alpha 5beta PC and 3beta 5beta PC, block by 2 µM 3alpha 5beta PS was dependent upon GABA concentration, depressing responses to 2 µM GABA (-90 mV) by 13 ± 3% and responses to 20 µM GABA by 58 ± 2%. Also like 3beta 5beta PC, 3alpha 5beta PS exhibited apparent voltage dependence, especially at low GABA concentrations. At +90 mV, block was increased from 13 ± 3% (-90 mV) to 30 ± 5% (N = 6). Because of the apparent use dependence of 3alpha 5beta PS block, the slight decrease in the effect of drug at pH 5.8 (Fig. 8D) may be related to the reduced amplitude of GABA responses at low pH. We found that pregnenolone sulfate (2 µM) block was also only slightly affected by pH 5.8 (91 ± 2% depression, N = 4 at pH 7.4, versus 76 ± 5% depression, N = 2 at pH 5.8). In addition, 3beta 5beta PS and dehydroepiandrosterone sulfate block were similarly weakly affected by pH (data not shown). These data are consistent with the idea that the effect of pH on carboxylated steroid block results primarily from the ionization state of the steroid rather than to sites on the receptor. These experiments also showed that only low micromolar concentrations of sulfated steroids are needed to match the degree of block given by the carboxylated steroids, suggesting that block by sulfated steroids is substantially more potent (~25 fold) than block by carboxylated steroids. In summary, these experiments suggest that both potentiation and block by carboxylated steroids are dramatically and inversely affected by pH and are consistent with the idea that the local environment of the steroid dramatically affects the steroid pK.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Novel Combination of Cellular Actions for 3alpha 5beta PC. The unique attribute of the neuroactive steroid 3alpha 5beta PC is the ability of this compound to potentiate GABAA receptor function and inhibit NMDA receptor function. This combination of cellular effects may enhance the clinical profile over previously characterized anesthetic neuroactive steroids. While block of NMDA receptors is perhaps sufficient to protect against various forms of glutamate-mediated toxicity (Michaelis, 1998), maximum block of NMDA receptors by neuroactive steroids is typically less than 100%. Therefore, the GABA-blocking actions of these same steroids may promote an increase in synaptic activity that at least partially undermines the direct effects on NMDA receptors. Likewise, GABA-potentiating actions should enhance the anesthetic properties of NMDA receptor blockers, because GABAA receptor potentiation and NMDA receptor block are the cellular actions most closely correlated with anesthetic properties (Franks and Lieb, 1994).

While 3alpha 5beta PC is a lead for developing neuroactive steroids with favorable clinical properties, several features of 3alpha 5beta PC may not be desirable or optimal for anticonvulsant, anesthetic, and neuroprotective properties. The most serious problem with this compound may be the blocking effect on GABAA receptors at high micromolar concentrations of drug. Therefore, this work focused on defining structural and functional aspects of carboxylated steroids at GABAA receptors. Block of GABAA receptors requires significantly higher concentrations of steroid than endogenous sulfated steroids, but these same high micromolar concentrations are required for activity at NMDA receptors. Given that 50 µM 3alpha 5beta PC produces no net change in IPSC time course but significantly dampens NMDA EPSCs (Fig. 1), 3alpha 5beta PC could be a better neuroprotective agent than previously reported C3 sulfates and hemiesters, all of which reportedly block GABAA receptors (Park-Chung et al., 1999). Acidosis produced during acute central nervous system insults such as anoxia would probably minimize GABAA block and maximize the potentiating effects of 3alpha 5beta PC (Figs. 7 and 8).

On the other hand, minimizing block of GABAA receptors while retaining or enhancing NMDA receptor block would probably produce a neuroactive steroid with better clinical utility. Given the resistance of GABAA receptor block to major changes in the stereochemistry or placement of the carboxylic acid (Figs. 3 and 5), it may prove difficult to eliminate block. Rather, ongoing work in our laboratories is aimed at defining and optimizing structural requirements for NMDA receptor block, which may prove a more fruitful strategy for improving the clinical usefulness of carboxylated steroids.

Action at GABAA Receptors: Site(s) of Action. Given that 3alpha 5beta PC both potentiates and blocks GABA receptors, it is worth considering current evidence for possible sites on the GABAA receptor that may mediate these actions. Potentiation by 3alpha 5beta PC exhibits clear stereoselectivity, whereas block does not. Likewise, concentration-response data at synapses (Fig. 1E) suggest that potentiation is most prominent at low micromolar concentrations of steroid, whereas block becomes apparent at concentrations >10 µM. Potentiation and block by 3alpha 5beta PC can also be temporally separated during voltage jumps to positive potentials (Fig. 3, A-F). Finally, low pH has opposite effects on block and potentiation, nearly eliminating block whereas enhancing potentiation. These results are all consistent with the hypothesis that potentiation and block occur at separate sites on the GABAA receptor. Previous work also suggests that positive modulators of GABAA receptors, such as (3alpha ,5alpha )-3-hydroxypregnan-20-one, act at a different GABAA receptor-associated site than blocking steroids such as pregnenolone sulfate (Zaman et al., 1992; Park-Chung et al., 1999). Unfortunately, specific potentiating and blocking sites on the GABAA receptor have thus far eluded identification, although a point mutation in transmembrane domain 2 dramatically reduces block by pregnenolone sulfate, perhaps by altering the transduction mechanism of block (Akk et al., 2001). Interestingly, it was previously shown that 3alpha 5beta PS and pregnenolone sulfate lack enantioselectivity for block of GABAA receptors, possibly suggesting the lack of a conventional chiral protein-ligand recognition site for pregnane and pregnene series blockers (Nilsson et al., 1998).

Potentiation by 3alpha 5beta PC and block by 3beta 5beta PC and lithocholic acid are pH-dependent. The apparent pK values for potentiation and block were 6.4 to 6.9. Because these values are approximately 1.5 to 2 units above the pK of lithocholic acid in water (Fini et al., 1987), we suggest that the pK of carboxylated steroids is significantly altered by the local environment in which the steroids act. The pK for many organic acids, including lithocholic acid, is increased from ~5 in water to ~8 in aqueous organic solvents (Fini et al., 1987). The pK of organic acids is also raised by association of the acid with membrane (Smejtek et al., 1987). Therefore, the local environment of the carboxylated steroids can dramatically affect the pK value and probably accounts for the high apparent pK values observed in the present study.

We found that potentiation effects at GABA receptors probably are mediated by the un-ionized form of 3alpha 5beta PC. Although potentiation by 3alpha 5beta PC was increased by decreased ionization, electroneutrality at C3 is not sufficient for potentiation. Another requirement seems to be that the substituent at C3 be a hydrogen bond donor (Phillipps, 1975). Consistent with this hypothesis, the methyl ester derivative of 3alpha 5beta PC was inactive at GABA receptors, and the amide derivative of 3alpha 5beta PC was an effective but pH-independent potentiator. The un-ionized form of 3alpha 5beta PC probably constitutes slightly less than 10% of the total steroid at physiological pH (assuming a pK of 6.4), and significant potentiation occurs at 5 µM total steroid. This suggests that protonated 3alpha 5beta PC is probably active at less than 500 nM, making the un-ionized form of 3alpha 5beta PC slightly lower in potency than other unsulfated neuroactive steroids that potentiate GABA responsiveness.

Given that potentiation and block by 3alpha 5beta PC probably occur through different sites (Park-Chung et al., 1999), future work will be aimed at structural modifications to 3alpha 5beta PC that maximize potentiation but minimize block. Ideally, we seek a compound for which only the un-ionized form will potently interact with GABA receptors but that retains block of NMDA receptors.

    Acknowledgments

We thank Joe Henry Steinbach and Gustav Akk (Washington University) for discussion and Robert Purdy (Scripps Research Institute) for generously supplying 3beta -hydroxy-5beta -pregnane-20-one sulfate.

    Footnotes

Received April 16, 2001; Accepted June 27, 2001

This work was supported by National Institute of Health Grant GM47969 (to C.F.Z., D.F.C., A.S.E.), a National Alliance for Research on Schizophrenia and Depression Young Investigator Award (to S.M.), a Klingenstein Foundation Grant (to S.M.), and a gift from the Bantly Foundation (to C.F.Z.).

Dr. Steven Mennerick, Ph.D., Department of Psychiatry, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8134, St. Louis, MO 63110. E-mail: menneris{at}psychiatry.wustl.edu

    Abbreviations

GABA, gamma -aminobutyric acid; NMDA, N-methyl-D-aspartate; C3, carbon 3; 3alpha 5beta PC, (3alpha ,5beta )-20-oxo-pregnane-3-carboxylic acid; IPSC, inhibitory postsynaptic current; 3alpha 5beta PS, 3alpha -hydroxy-5beta -pregnan-20-one sulfate; 3alpha 5beta PA, (3