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The Journal of Neuroscience, November 5, 2003, 23(31):10013-10020
 Previous Article | Next Article 
Cellular/Molecular
The Contraceptive Agent Provera Enhances GABAA Receptor-Mediated Inhibitory Neurotransmission in the Rat Hippocampus: Evidence for Endogenous Neurosteroids?
Delia Belelli and
Murray B. Herd
Department of Pharmacology and Neuroscience, University of Dundee, Ninewells Hospital and Medical School, Ninewells Hospital, Dundee, DD1 9SY United Kingdom
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Abstract
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Neurosteroids typified by 5 -pregnan-3-ol-20-one (53) have emerged as the most potent endogenous positive modulators of the GABAA receptor, the principal mediator of fast inhibitory transmission within the CNS. Neurosteroids can be synthesized de novo in the brain in levels sufficient to modulate GABAA receptor function and, thus, might play an important physiological-pathophysiological role. Indirect support for this proposal comes from the observation that neurosteroid action is region and neuron selective. However, the mechanism(s) that imparts specificity of action remains primarily elusive. Although neurosteroids are relatively promiscuous toward different GABAA receptor isoforms, the contribution of local neurosteroid metabolism has been relatively unexplored. Here, we investigate the role of neurosteroid metabolism by using electrophysiological techniques to compare the actions of 53 and its metabolically stable synthetic analog ganaxolone on inhibitory neurotransmission in CA1 and dentate gyrus neurons. Furthermore, we evaluate the contribution of a key enzyme in neurosteroid metabolism [i.e., 3-hydroxysteroidoxidoreductase (3-HSOR)] to the inactivation of endogenous, or exogenously applied 53. We show that low concentrations of ganaxolone, but not of 53, enhance inhibitory transmission in dentate gyrus, whereas both steroids are similarly effective in CA1 neurons. Furthermore, inhibition of 3-HSOR by the contraceptive agent Provera results in enhanced synaptic and extrasynaptic GABAA receptor-mediated inhibition in the dentate gyrus but not in the CA1 region. Collectively, these findings advocate a crucial role for local steroid metabolism in shaping GABAA receptor-mediated inhibition in a regionally dependent manner and suggest a novel action by the contraceptive agent on inhibitory centers in the CNS.
Key words: neurosteroid; inhibitory synaptic transmission; GABAA receptor; tonic current; hippocampus; patch-clamp
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Introduction
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A number of independent investigations have established that certain endogenous neurosteroids can potently and selectively enhance the action of the inhibitory neurotransmitter GABA at the GABAA receptor (Paul and Purdy, 1992 ; Lambert et al., 2001; Smith, 2002). Their behavioral repertoire is consistent with an enhancement of inhibition (i.e., they display anxiolytic, anticonvulsant, and analgesic activity) and, at higher doses, they are hypnotic and anesthetic (Belelli et al., 1990; Lambert et al., 1995). Neuroactive steroids might also be involved in the physiological and pathophysiological regulation of neuronal inhibition. Physiological levels of the most potent neurosteroid [i.e., 5-pregnan-3-ol-20-one (53)] fall within the range of concentrations ( 10 nM) shown in vitro to enhance GABAA receptor function (Belelli et al., 1996a,b, 2002). Furthermore, neurosteroids can be synthesized de novo in the brain independent of peripheral sources both in glia and neurons. Thus, 53 can be synthesized from progesterone via the sequential action of 5-reductase and 3-hydroxysteroidoxidoreductase (3-HSOR). Interestingly, in contrast to 5-reductase, 3-HSOR is involved in both the synthesis and degradation of 53 because, depending on whether the cytosolic or membrane-bound isoform prevails, it can either reduce 5-dihydroprogesterone (5-DHP) to 53 or oxidize 53 back to 5-DHP, respectively (Mellon and Vaudry, 2001). The fact that the brain expression pattern and activity of the enzymes responsible for neurosteroid synthesis and degradation will selectively impact on GABAA receptor-mediated inhibition is supported by the following observations: (1) the activity of the enzymes involved in neurosteroid metabolism exhibits regional variations (Mellon and Vaudry, 2001), (2) administration of finasteride, an inhibitor of 5-reductase, causes a dramatic decrease in rat brain 53 levels and concomitant changes in behavior consistent with the loss of an inhibitory tone (Matsumoto et al., 1999; Pinna et al., 2000), and (3) in animal models of psychiatric illness, inhibitory dysfunction appears to be associated with a loss of 5-reductase activity (Dong et al., 2001). Findings from this and other laboratories have suggested that neurosteroid modulation of inhibitory transmission is neuron specific, a heterogeneity that might be conferred in part by the isoform of the GABAA receptor and post-translational modifications of the receptor, including phosphorylation (Cooper et al., 1999; Belelli et al., 2002; Harney et al., 2003; Koksma et al., 2003). However, the contribution of local neurosteroid metabolism to neurosteroid action on GABAA receptor-mediated neurotransmission has received little attention. Thus, we compared the actions of 53 and its metabolically stable synthetic analog ganaxolone (3 -methyl-3-ol-5-pregnan-20-one) in hippocampal CA1 pyramidal neurons and dentate gyrus (DG) granule cells. Additionally, we evaluated the contribution of 3-HSOR activity to the inactivation of endogenous or exogenously applied 53 by using two structurally distinct inhibitors of 3-HSOR, the anti-inflammatory agent indomethacin, and the commonly used contraceptive progestin, Provera. Interestingly, Provera is also effective in the treatment of catamenial epilepsy (Newmark and Penry, 1980). The results presented here advocate a crucial role for local steroid metabolism in shaping GABAA receptor-mediated inhibition in a regionally dependent manner. Furthermore, the findings with Provera imply that the contraceptive agent might alter the function of inhibitory centers in the CNS.
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Materials and Methods
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Recordings from rat hippocampal slices
Slice preparation. Hippocampal slices were prepared from Sprague Dawley rats of either sex [postnatal day (P) 16-P24] according to standard protocols described previously (Reynolds et al., 2003). Animals were killed by cervical dislocation in accordance with Schedule 1 of the United Kingdom Government Animals (Scientific Procedures) Act, 1986. The brain was rapidly removed and placed in oxygenated ice-cold artificial CSF (aCSF) solution containing the following (in mM): 225 sucrose, 2.95 KCl, 1.25 NaH2PO4, 26 NaHCO3, 0.5 CaCl2, 10 MgSO4, 10 Glucose, 1 ascorbic acid, and 3 pyruvic acid bubbled with 95% O2/5% CO2 to give a pH of 7.4 (330-340 mOsm). The tissue was maintained in ice-cold aCSF while 300 µm horizontal slices were cut using a Vibratome (Intracel, Royston, UK). The slices were placed in an incubation chamber, consisting of a beaker and a nylon mesh-covered plastic ring on which the slices lay, filled with circulating, oxygenated, extracellular solution containing the following (in mM): 126 NaCl, 2.95 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 10 D-glucose, and 2 MgCl2, pH 7.4, when bubbled with 95% O2/5% CO2 (300-310 mOsm). Slices were incubated at 32°C for 1 hr and subsequently allowed to cool at room temperature before being used for recordings.
Electrophysiology. Whole-cell patch-clamp recordings were made at 35°C from hippocampal CA1 pyramidal neurons and dentate granule cells visually identified with a Zeiss 2FS (Zeiss, Welwyn Garden City, UK) or Olympus BX51 (Olympus, Southall, UK) microscope equipped with differential interference contrast-infrared optics. Patch pipettes were prepared from thick-walled borosilicate glass (Garner Glass, Claremont, CA) and had open tip resistances of 3-5 M when filled with an intracellular solution that contained the following (in mM): 140 CsCl, 10 HEPES, 10 EGTA, 2 Mg-ATP, 1 CaCl2, 5 QX-314, pH 7.3, with CsOH, 295-305 mOsm. Miniature IPSCs (mIPSCs) were recorded using an Axopatch 1D or Axopatch 200B amplifier (Axon Instruments, Foster City, CA) at a holding potential of -60 mV in an extracellular recording solution (see above) also containing 2 mM kynurenic acid (Sigma, Poole, UK) and 0.5 µM tetrodotoxin (TTX; Tocris, Bristol, UK) to block ionotropic glutamate receptors and action potentials, respectively. To ensure a reliable detection of the tonic current in dentate granule cells, experiments investigating the influence of steroid or Provera were performed in the absence of TTX on slices previously incubated in the presence of the GABA transaminase inhibitor, 50 µM vigabatrin (Sigma) for 2-4 hr (Yeung et al., 2003). Series resistance ranged from 6-18 M and was compensated up to 80%. In each case, currents were sampled at 10 kHz and filtered at 2 kHz using an 8-pole low-pass Bessel filter.
Drug application. 53, ganaxolone, indomethacin, and Provera were prepared as concentrated (1000x) stock solutions in DMSO while pentobarbital and bicuculline methobromide (10-2 M) were dissolved in water. These stock solutions were diluted in the extracellular solution to the desired concentration. The final maximum DMSO concentration (0.2%) had no effect on any of the mIPSC parameters or the tonic current. Preliminary time course experiments revealed that the steroid and 3-HSOR inhibitors effect was maximum after 5-8 min of exposure and stable afterward up to 30 min. Thus, all modulatory agents were applied via the perfusion system (2-4 ml/min) and allowed to infiltrate the slice for a minimum of 10 min before recordings were acquired. With the exception of 53 and ganaxolone, which were generous gifts from Dr. K. Gee (University of California, Irvine, CA), all drugs tested were obtained from Sigma.
Data analysis. Data were recorded onto a digital audiotape (DAT) using a Bio-Logic (Claix, France) DTR 1200 recorder and analyzed off-line using the Strathclyde Electrophysiology Software, WinEDR/WinWCP (courtesy of Dr. J. Dempster, University of Strathclyde, Glasgow, UK). Individual mIPSCs were detected using a -4 pA amplitude threshold detection algorithm and visually inspected for validity. Accepted events were analyzed with respect to peak amplitude, 10-90% rise time, charge transfer, and time for events to decay by 50% (T50) and 90% (T90). To minimize the contribution of dendritically generated currents, which are subject to the unquantifiable effects of cable filtering, analysis was limited to those events that fell within the limits of a Gaussian distribution that describes the peak of the rise-time histogram. Analysis of rise-time histograms generally revealed a skewed distribution with a clear peak below 1 msec. Miniature IPSCs were fitted with either one or two exponentials given by the equations: y(t) = A · e(-t/ ), for mono-exponential mIPSCs; and y(t) = A1 · e(-t/1) + A2 · e(-t/2) for bi-exponential mIPSCs, where A is amplitude, t is time, and is the decay time constant. An improvement of the fit by two exponentials compared with one resulted in a reduction in the SD of the residuals and was confirmed by use of the F-test. To compare mono-exponentially versus bi-exponentially decaying events recorded from each cell, a weighted decay time constant (w) was also calculated for those mIPSCs exhibiting a bi-exponential decay (w = 1 · P1 + 2 · P2, where 1 and 2 are the decay time constants of the first and second exponential functions, and P1 and P2 are the proportions of the current amplitude described by each component.). Individual values derived from mono-exponentially decaying events and w values were averaged together to give a cumulative value. Tonic current amplitude was calculated as the difference between the holding current before and after application of 30 µM bicuculline methobromide (Brickley et al., 1996).
All results are reported as the arithmetic mean ± SEM. The large sample approximation of the Kolmogorov-Smirnoff (KS) test (SPSS software; SPSS, Chicago, IL) was used to compare the distribution of the mIPSCs parameters. Statistical significance of mean data was assessed with the unpaired Student's t test or repeated measures ANOVA post hoc followed by the Newman-Keuls test as appropriate, using the SigmaStat (SPSS) software package.
Recordings from Xenopus laevis oocytes. Xenopus laevis oocytes (stage V-VI) were isolated as described previously (Belelli et al., 1996a) and injected into their cytoplasm with 20-40 nl of cRNA transcripts (1 mg/ml) prepared from linearized human GABAA 1, 3, and  2 cDNA according to standard protocols. Two to twelve days after injection, recordings were performed on such oocytes voltage-clamped at -60 mV with an Axoclamp 2A (Axon Instruments) in the twin-electrode voltage-clamp mode. The voltage and current-passing electrodes were filled with 3 M KCl and had resistances of 0.8-2 M when measured in the recording solution (frog Ringer). The oocytes were continually superfused with frog Ringer containing the following (in mM): 120 NaCl, 1.8 CaCl2, 2 KCl, and 5 HEPES, pH 7.4, with NaOH, at the rate of 7-10 ml/min. Membrane current responses were low-pass filtered at 200 Hz, recorded onto DATs via a Bio-Logic DAT recorder (DTR1200), and simultaneously displayed on a Lectromed (Letchworth, UK) multitrace two-pen recorder. All drugs were applied via the superfusion system. The effects of indomethacin and Provera were assessed by using a concentration of GABA producing a half-maximal response (i.e., EC50). Indomethacin and Provera were prepared as concentrated stock (1000x) in DMSO and diluted to the desired concentration in frog Ringer. The final maximum DMSO concentration (0.1%) had no effect on the control GABA-evoked response. Drugs were preapplied for 40-60 sec before being coapplied with the appropriate GABA concentration. Data were expressed as a percentage of the control response and statistically analyzed by repeated measures ANOVA.
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Results
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As a prelude to investigate the role of neurosteroid metabolism in shaping GABAergic synaptic transmission, we first characterized the properties of GABAA receptor-mediated mIPSCs recorded at 35°C from hippocampal pyramidal CA1 and DG neurons. Such currents were reversibly blocked by the competitive GABAA receptor antagonist bicuculline (30 µM; data not shown). As illustrated in Table 1, CA1 and DG mIPSC parameters were similar and in agreement with previous studies on rats of similar age (Hollrigel and Soltesz, 1997; Harney et al., 2003).
The effect of 53 and ganaxolone on mIPSCs recorded from CA1 and DG neurons
Previous investigations have suggested that GABAA receptors mediating synaptic transmission in hippocampal DG but not CA1 neurons are insensitive to the action of low ( 100 nM) concentrations of certain neurosteroids [e.g., 5-tetrahydro-deoxycorticosterone (5-THDOC) or 5-pregnan-3-ol-20-one], although higher steroid concentrations did enhance inhibitory transmission (Cooper et al., 1999; Harney et al., 2003). We, and others, have shown that the subunit composition and phosphorylation of the GABAA receptor or associated proteins influence neurosteroid action (Puia et al., 1993; Fancsick et al., 2000; Belelli et al., 2002; Harney et al., 2003; Koksma et al., 2003). However, these are unlikely to fully account for the differential neurosteroid sensitivity of dentate granule cells compared with CA1 neurons (see Discussion). An alternative, yet not mutually exclusive, explanation might lie in local steroid metabolism, which could blunt the steroid action in the dentate gyrus but not in the CA1 region. To test this hypothesis, we investigated and compared the actions of the most potent neurosteroid 53 and its metabolically stable synthetic analog ganaxolone on GABAA receptor-mediated mIPSCs recorded from CA1 and DG neurons. As shown in Figure 1, A and B, low concentrations (100 nM) of 53 significantly enhanced inhibitory transmission in CA1 neurons (i.e., the steroid prolonged the synaptic current decay of GABAA receptor-mediated mIPSCs in all cells tested; p < 0.05; KS test). Thus, 30 and 100 nM 53 significantly increased by 11 ± 3% (n = 5; p < 0.05) and 25 ± 5% (n = 7; p < 0.01), respectively (Table 2), whereas no significant effect was observed on the peak amplitude (data not shown). The synthetic derivative ganaxolone was similarly effective in CA1 neurons (Fig. 1C,D), with 30 and 100 nM of the steroid producing a significant increase of (p < 0.01 for both steroid concentrations) (Table 2) and no significant effect on the peak amplitude (data not shown). In contrast, when 53 action was investigated in the dentate gyrus, only 1 of 7 and 3 of 7 cells were sensitive to 30 and 100 nM of the steroid, respectively (KS test; p < 0.05). Overall, the changes in values were not significantly different from control for either concentration (Fig. 2A,B, left; Table 2) (p > 0.05 for both concentrations). However, 30 and 100 nM ganaxolone prolonged the mIPSCs synaptic current decay of every DG neuron tested (n = 6-7; p < 0.05; KS test) (Fig. 2A,B, right; Table 2).
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Figure 1. The effect of 53 and ganaxolone (GNX) on the decay of mIPSCs recorded from CA1 neurons. A, A cumulative probability plot of the decay of all mIPSCs, expressed as the cumulative time constant (see Materials and Methods), recorded from an exemplar CA1 neuron before and after the application of 30 (left) and 100 nM (right) 53. The rightward shift of this relationship induced by either steroid concentration indicates that all mIPSCs recorded from this cell were sensitive to the neurosteroid. The insets illustrate the normalized ensemble average of all mIPSCs from the same CA1 neurons before and after application of 30 (left) and 100 nM (right) 53. Calibration: 20 pA (left), 10 pA (right), 10 msec. B, Examples of individual mIPSC traces recorded from the same CA1 neuron before and after the application of 100 nM 53. C, A cumulative probability plot of the decay of all mIPSCs, expressed as the cumulative time constant (see Materials and Methods), recorded from an exemplar CA1 neuron before and after the application of 30 (left) and 100 nM (right) ganaxolone. The rightward shift of this relationship induced by either steroid concentration indicates that all mIPSCs recorded from this cell were sensitive to this steroid. The insets illustrate the normalized ensemble average of all mIPSCs from the same CA1 neurons before and after application of 30 (left) and 100 nM (right) ganaxolone. Calibration: 10 pA, 10 msec. D, Examples of individual mIPSC traces recorded from the same CA1 neuron before and after the application of 100 nM ganaxolone.
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The effect of inhibitors of 3-hydroxysteroidoxidoreductase on mIPSCs recorded from CA1 and DG neurons
It is clearly established that although the reduction of progesterone to 5-DHP by 5-reductase is irreversible, the reduction of the latter to 53 by 3-HSOR can reverse to the inactive precursor (i.e., 5-DHP) (Mellon and Vaudry, 2001). Thus, we hypothesized that the differential sensitivity of DG granule cells to the action of 53 versus ganaxolone may be accounted for by local steroid metabolism, that is, the endogenous progesterone metabolite would be oxidized back to the inactive precursor 5-dihydroprogesterone, whereas ganaxolone, by virtue of the methyl group in the 3 position, would not (Carter et al., 1997). Thus, to test this hypothesis and investigate the contribution of local steroid metabolism to the selectivity of neurosteroid action, we used indomethacin (10 µM) to inhibit the activity of 3-HSOR. This concentration equates to five times the calculated IC50 for indomethacin inhibition of rat brain 3-HSOR and, thus, is expected to completely block the activity of this enzyme (Penning et al., 1985). In a series of preliminary experiments, 10 µM indomethacin was devoid of action at recombinant 132 GABAA receptors expressed in Xenopus laevis oocytes (response, 92 ± 3% of control; n = 4; data not shown). However, when the enzyme inhibitor was tested on mIPSCs recorded from DG neurons, the synaptic current decay (i.e., ) was significantly prolonged by 20 ± 4% (n = 7; p < 0.05) (Fig. 3A, left; Table 3). To validate the specificity of this effect, we also used the structurally distinct contraceptive agent Provera, which is reported to inhibit rat brain 3-HSOR with an  10-fold greater potency than indomethacin (Penning et al., 1985). In common with indomethacin, Provera (1 µM) was devoid of action at recombinant 132 GABAA receptors expressed in Xenopus laevis oocytes (response, 90 ± 5% of control; n = 4; data not shown) but significantly prolonged the synaptic current decay of mIPSCs recorded from DG neurons by 24 ± 5% (n = 7; p < 0.05) (Fig. 3A, right; Table 3). The increase in produced by 1 µM Provera was not significantly different from that caused by 10 µM indomethacin (p > 0.05) (Table 3). Furthermore, in the presence of 1 µM Provera, 100 nM 53 produced a significant increase of in addition to that produced by Provera or 53 alone (p < 0.05) (Table 3). Therefore, these results suggest that when metabolic inactivation is prevented, an endogenous neurosteroid tone is revealed, which is sufficient to modulate synaptic inhibition. Thus, these findings are consistent with the view that neurosteroid metabolism influences GABAA receptor-mediated synaptic transmission in the dentate gyrus and, in addition, the selectivity of action of administrated neuroactive steroids.
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Table 3. The effect of indomethacin and Provera on the decay time constant of mIPSCs recorded from CA1 and DG neurons
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The observation that, in contrast to the situation of the dentate gyrus, 53 and ganaxolone are similarly effective at prolonging GABAA receptor-mediated mIPSCs in CA1 neurons suggests that 3-HSOR activity might display regional differences. To test this hypothesis, we evaluated and compared the actions of indomethacin and Provera on mIPSCs recorded from CA1 neurons. As illustrated in Table 3 and Figure 3B, 10 µM indomethacin had no significant effect (increase, 7 ± 4%; n = 5; p > 0.05) on CA1 mIPSCs, and 1 µM Provera produced only a modest but significant increase of (increase, 8 ± 2%; n = 8; p < 0.05). Furthermore, the effects of these 3-HSOR inhibitors in CA1 were significantly different from those observed in the DG (p < 0.05 for both indomethacin and Provera in CA1 vs dentate gyrus) (Table 3). These results imply that 3-HSOR activity differs between CA1 and dentate gyrus and, hence, influences the ability of administrated neuroactive steroids to modulate GABAA receptor-mediated neurotransmission in a manner that is neuron selective (see Discussion).
The effect of 53, ganaxolone, and Provera on the extrasynaptic GABAA receptor-mediated tonic conductance
A number of reports have provided compelling evidence for the presence in the dentate gyrus of an extrasynaptic GABAA receptor-mediated tonic conductance, which has been suggested to be mediated by receptors composed of 4,x and  subunits (Mody, 2001; Nusser and Mody, 2002; Stell and Mody, 2002). Recently, we and others have shown that this receptor combination is exquisitively sensitive to the actions of neurosteroids and to those of a number of therapeutically used drugs, which include the anesthetic agents etomidate and pentobarbital but not benzodiazepines (Belelli et al., 2002; Brown et al., 2002; Wohlfarth et al., 2002). Thus, we reasoned that if 3-HSOR enzymatic activity in the DG prevents modulation of synaptic GABAA receptors by exogenously applied 53, this may additionally hinder modulation of the extrasynaptic GABAA receptor-mediated tonic conductance by 53 but not by ganaxolone. Thus, in agreement with previous findings (Stell and Mody, 2002), application of bicuculline (30 µM) unmasked a tonic current of 32 ± 4 pA (n = 17). More importantly, 100 nM ganaxolone enhanced the tonic current in four of five cells tested from 21 ± 4 to 47 ± 7 pA (increase, 136 ± 28%; p < 0.05) (Fig. 4). In contrast, the tonic current from only one of five cells was sensitive to 53 action with an increase of 147%. Overall, the tonic current in the presence of 100 nM 53 was not significantly different from that calculated in its absence (control, 32 ± 8 pA; plus 100 nM 53, 39 ± 8 pA; p > 0.05; n = 5) (Fig. 5). However, block of 3-HSOR by 1 µM Provera resulted in a significant increase of the tonic current from 40 ± 9 to 72 ± 18 pA (increase, 74 ± 8; p < 0.05; n = 5) (Fig. 6). The action of this contraceptive was blocked by the subsequent application of 30 µM bicuculline, thus demonstrating that the effects of Provera are GABAA receptor-mediated. Collectively, these results demonstrate that metabolic inactivation of the endogenous neurosteroid tone in the dentate gyrus modulates inhibitory neurotransmission at both synaptic and extrasynaptic loci.
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Figure 4. The effect of ganaxolone on the tonic current recorded from DG neurons. A, The tonic current, calculated as the difference between the holding current in the presence and absence of 30 µM bicuculline (see Materials and Methods), recorded from an exemplar DG neuron is enhanced by the application of 100 nM ganaxolone, and the steroid effect is blocked by the subsequent application of 30 µM bicuculline. The dashed line indicates the holding current (pA) under control conditions. Calibration: 50 pA, 5 sec. B, Corresponding all-point histograms illustrate the amplitude of the holding current under control conditions (CTRL) in the presence of 100 nM ganaxolone (GNX) and after the application of 30 µM bicuculline (BIC). C, Bar graph summarizing the enhancement of the tonic current induced by 100 nM ganaxolone in five DG neurons.
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Figure 6. The effect of Provera on the tonic current recorded from DG neurons. A, The tonic current, calculated as the difference between the holding current (pA) in the presence and absence of 30 µM bicuculline (see Materials and Methods), recorded from an exemplar DG neuron is enhanced by the application of 1 µM Provera, and this effect is blocked by the subsequent application of 30 µM bicuculline. The dashed line indicates the holding current under control conditions. Calibration: 50 pA, 5 sec. B, Corresponding all-point histograms illustrate the amplitude of the holding current under control conditions (CTRL) in the presence of 1 µM Provera (PRO) and after the application of 30 µM bicuculline (BIC) C, Bar graph summarizing the enhancement of the tonic current induced by 1 µM Provera in five DG neurons.
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Discussion
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The action of 53 and ganaxolone on GABAA receptor-mediated inhibition in CA1 and dentate gyrus
The aim of the present investigation was twofold: (1) to evaluate the contribution of neurosteroid metabolism to the differential steroid sensitivity of neuronal GABAA receptors, and (2) to assess whether physiologically neurosteroid metabolism exerts a permissive role to shape GABAA receptor-mediated neurotransmission in a regionally dependent manner. To address the first objective, we compared and contrasted the action of the potent endogenously occurring progesterone metabolite 53 and the metabolically stable 53 analog, ganaxolone, on GABAA receptor-mediated mIPSCs recorded from CA1 and dentate granule cells. The findings presented here show that low concentrations of both 53 and ganaxolone are similarly effective at prolonging mIPSCs recorded from CA1 neurons. These results are consistent with the observation that 53 and ganaxolone are both equally effective and potent in enhancing the function of both recombinant and native GABAA receptors (Belelli et al., 1996a, b; Carter et al., 1997). In contrast, in the dentate gyrus, both synaptic and extrasynaptic GABAA receptors are sensitive to low concentrations of the synthetic analog ganaxolone but not to 53. The relative insensitivity of DG synapses to 53 is in agreement with previous reports on low concentrations (100 nM) of either the 5-isomer or 5-THDOC in brain slices prepared from rats of comparable age (Cooper et al., 1999; Harney et al., 2003). Proposed reasons for this relative insensitivity include receptor subunit composition and phosphorylation. Of the subunits expressed in dentate gyrus and CA1 that may impact on neurosteroid action (Nusser et al., 1996; Brooks-Kayal et al., 2001), the 1 subunit and the 4 and -containing receptor assembly emerge as putative candidates to impart increased neurosteroid sensitivity. In contrast, receptors incorporating the 2 subunit are significantly less sensitive to physiologically relevant concentrations (100 nM) of 53 (Belelli et al., 2002; Brown et al., 2002). However, recombinant GABAA receptors containing any of these subunits do not discriminate between 53 and ganaxolone (Carter et al., 1997; Mascia et al., 2002; D. Belelli, unpublished observations). In addition, 4 and -containing recombinant receptors are exquisitively sensitive to steroid action.
Here, however, we demonstrate that extrasynaptic GABAA receptors in the dentate gyrus are relatively insensitive to 53 and, yet, such receptors are thought to comprise the 4 and subunits (Mody, 2001). Thus, it seems highly unlikely that the receptor subunit composition might explain the differential sensitivity of DG cells to 53 and ganaxolone. Recently, phosphorylation has been shown to impact on neurosteroid enhancement of synaptic inhibition (Fancsick et al., 2000; Vicini et al., 2002; Harney et al., 2003; Koksma et al., 2003). Although the precise mechanism by which such regulation occurs remains to be determined, it appears improbable that phosphorylation might selectively influence the actions of 53 but not those of ganaxolone. Collectively, these observations strongly implicate local metabolism in the differential sensitivity of DG cells to the endogenous and synthetic steroid. In rat brain, 53 can be metabolized by the following different pathways: (1) it can be reduced at the 20 position to the potent GABAA receptor-active neurosteroid 5-pregnan-3,20-diol (Belelli et al., 1996b), (2) it can be sulfated at the 3-OH group, and (3) it can be oxidized back to the 53 precursor (i.e., 5-DHP). The latter two metabolites are devoid of action at GABAA receptors with the conversion of 53 to 5-DHP being the most important pathway at least in the brain (Korneyev et al., 1993; Dong et al., 2001). In contrast, ganaxolone, by virtue of the methyl group in the 3 position, potentially can only be converted to the 20 hydroxy GABAA receptor-acting metabolite or possibly the 3-sulfate ester. Contrary to 53, the anticonvulsant action of ganaxolone in both animals and humans is relatively long lasting (Carter et al., 1997; Monaghan et al., 1997). Thus, although a detailed pharmacokinetic profile of ganaxolone in the rodent brain is not available, either the steroid is in the main metabolically stable or the primary metabolites must be active at the GABAA receptor.
The role of 3-HSOR
The findings discussed above suggest that local neurosteroid metabolism can impart selectivity to the action of administrated steroids at the GABAA receptor. Furthermore, they imply that local metabolism of endogenous neurosteroids influences GABAA receptor-mediated inhibition in a neuron-specific manner. To validate this hypothesis, we blocked the activity of 3-HSOR in both CA1 and DG neurons with indomethacin and confirmed the specificity of this effect with a more potent, structurally diverse inhibitor of 3-HSOR (i.e., Provera). Although both ligands display additional pharmacological actions (e.g., indomethacin inhibits arachidonate cyclo-oxygenase, whereas Provera is active at cytosolic progesterone receptors) (Goodman et al., 2001), such actions are unlikely to account for the rapid prolongation of the mIPSC decay or enhancement of GABAA receptor-mediated extrasynaptic tonic conductance observed in dentate gyrus. In addition, neither agent exhibits a direct positive modulatory action on recombinant GABAA receptors.
The reaction catalyzing the conversion of 5-DHP to 53 is established as reversible (Mellon and Vaudry, 2001). Specifically, Li et al. (1997) have identified in rat brain two isoforms of 3-HSOR: one subtype that is cytosolic and primarily catalyzes the reaction in the reductive direction (i.e., leading to the formation of 53), and another isoform that is membrane-bound and catalyzes the reaction preferentially in the oxidative direction (i.e., leading to the formation of 5-DHP). Interestingly, when these two enzymatic activities were examined across the rodent brain, a differential regional distribution became apparent, and the dentate gyrus in particular was shown to have a high activity of the membrane-bound 3-HSOR isoform (Li et al., 1997). We speculate that in the dentate gyrus, the membrane-bound isoform of 3-HSOR regulates the endogenous levels of 53. This proposal is consistent with previous reports showing that conversion of nanomolar concentrations of 53 and 5-THDOC to 5-DHP and 5-deoxycorticosterone, respectively, occurs in rat brain (Purdy et al., 1991). Furthermore, 3H-53 is rapidly metabolized to 5-DHP in a neuroblastoma cell line, and this conversion can be blocked by indomethacin (Rupprecht et al., 1993). Whether the locus of enzyme action is intracellular remains to be determined. From the evidence presented here, it is argued that 3-HSOR activity in dentate gyrus is sufficient to prevent modulation of neuronal synaptic and extrasynaptic inhibition by relatively low concentrations of exogenously applied 53. Furthermore, the results with either indomethacin or Provera suggest that if not for metabolic inactivation, endogenous dentate gyrus levels of 53 would be high enough to enhance both synaptic and extrasynaptic GABAA receptor-mediated inhibition. Interestingly, the magnitude of the steroid effect on the decay of mIPSCs recorded from the dentate gyrus when 3-HSOR activity is blocked is similar to that produced by the same steroid concentration at CA1 synapses in the absence of inhibition of 3-HSOR. These findings, coupled with the observation that inhibition of 3-HSOR activity in CA1 has little if any effect on synaptic inhibition, suggest that the naturally occurring levels of 53 are low in both CA1 and dentate gyrus but for different reasons. In the dentate gyrus, local 53 is metabolically inactivated by 3-HSOR, whereas in CA1, the steroid levels are maintained low by other means, possibly because of a reduced synthesis.
To our knowledge, these results provide the first demonstration that 3-HSOR may shape neuronal inhibition mediated by both synaptic and extrasynaptic GABAA receptors in a neuron-specific manner. In addition, this enzyme influences the action of exogenously applied 53. Such findings are consistent with the recent report that IPSCs recorded from cortical neurons of in vitro brain slices obtained from mice injected systemically with a 5-reductase inhibitor (i.e., SKF 105111) display faster decay kinetics than their vehicle-injected controls (Puia et al., 2003). Collectively, these observations imply that physiological and pathophysiological alterations of neurosteroid anabolic or catabolic machinery might selectively alter neuronal inhibition. In support of this view, in a mouse model of psychiatric disorders, abnormally low levels of 53 are positively correlated with a reduced 5-reductase activity (Dong et al., 2001). It is tempting to speculate that some human neurological and psychiatric illnesses associated with inhibitory dysfunction and abnormal levels of GABAA receptor-acting neurosteroids (e.g., catamenial epilepsy and postpartum dysphoria) might also be characterized by regionally dependent alterations of the steroid metabolic machinery.
In summary, we demonstrated that selectivity of neurosteroid action at inhibitory GABAergic synaptic and extrasynaptic sites is influenced by local steroid metabolism in a neuron-specific manner. In this regard, we speculate that the ability of the contraceptive agent Provera to inhibit 3-HSOR might have important neurological implications on the long-term use of this progestin and the action of androgen and progesterone derivatives on inhibitory centers in the CNS. It is intriguing that administration of medroxyprogesterone (i.e., Provera) has long been known to be therapeutically beneficial for the treatment of catamenial seizures but not of noncatamenial epilepsy (Newmark and Penry, 1980). These observations highlight 3-HSOR as a potential novel target for the development of new therapeutics in the treatment of catamenial epilepsy. Importantly, these discoveries provide the starting point to investigate the role played by specific enzymes involved in neurosteroid metabolism in pathophysiological conditions associated with inhibitory dysfunction.
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Footnotes
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Received July 30, 2003;
revised September 12, 2003;
accepted September 16, 2003.
This work was supported by the Medical Research Council (MRC), Epilepsy Research Foundation, Anonymous Trust, and Commission of the European Communities Programme Quality of Life and Management of Living Resources, QLK1-CT-2000-00179. D.B. is an MRC Senior Fellow. We thank Dr. John Dempster for help and useful comments on the miniature IPSCs analysis.
Correspondence should be addressed to Delia Belelli, Department of Pharmacology and Neuroscience, University of Dundee, Ninewells Hospital and Medical School, Ninewells Hospital, Ninewells Avenue, Dundee, DD1 9SY United Kingdom. E-mail: d.belelli{at}dundee.ac.uk.
Copyright © 2003 Society for Neuroscience 0270-6474/03/2310013-08$15.00/0
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Abstract
Introduction
