Introduction

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Neurosteroids, according to the denomination proposed in 1981 by Baulieu (1981) and largely followed thereafter, are steroid hormones that accumulate in the brain independently of endocrine sources and can be synthesized from sterol precursors in nervous cells. Neurosteroids include progesterone, 5-pregnane-3-ol-20-one (allopregnanolone), pregnenolone, dehydroepiandrosterone (DHEA), or their respective sulfate esters (pregnenolone sulfate, DHEAS). Apart from their classical genomic effects, neurosteroids modulate several neurotransmission systems, in an excitatory or inhibitory way. Pregnenolone sulfate and DHEAS act as excitatory neurosteroids, since they antagonize the activation of -aminobutyric acid type A (GABA_A) receptors (Majewska and Schwartz, 1987; Majewska et al., 1988), whereas they potentiate the activation of the N-methyl-D-aspartate-type of glutamatergic receptors (Wu et al., 1991; Irwin et al., 1992, 1994; Maione et al., 1992; Bowlby, 1993). Some other neurosteroids, including progesterone or allopregnanolone, act as inhibitory neurosteroids, being very potent agonists of GABA_A receptors (Smith, 1991). As a consequence, neuro steroids are involved in several physiopathological events, such as response to stress, depression, anxiety, sleep, epilepsy, and memory formation (for reviews, see Schumacher et al., 1997; Maurice et al., 1999). Actually, the importance of neurosteroids in depression has been shown in several clinical studies. The concentration of allopregnanolone and pregnenolone in the cerebrospinal fluid of patients with major depression was lower than in control subjects (George et al., 1994; Uzunova et al., 1998). Mood-disordered subjects, who were clinically depressed, had lower cerebrospinal fluid pregnenolone as compared with healthy volunteers (George et al., 1994). It has been shown that treatment with antidepressant corrected the unbalance of neuroactive steroids observed in depressive patients (Romeo et al., 1998). One study has already reported a benefic effect of neurosteroids as antidepressant: patients with major depression and low basal plasma DHEA were openly administered DHEA, and depression ratings significantly improved during the time of the treatment (Wolkowitz et al., 1997). A double blind clinical study recently confirmed these data (Wolkowitz et al., 1999).

The ₁-receptor represents a unique binding site in the central nervous system and peripheral organs, distinct from any other known transmitter receptor. The ₁-receptor ligands exert a potent neuromodulation on excitatory neurotransmitter systems, including the noradrenergic, glutamatergic, and cholinergic systems (Matsuno et al., 1993; Bergeron et al., 1995; Gonzalez-Alvear and Werling, 1995a,b; Monnet et al., 1996). At the behavioral level, selective ₁-receptor ligands are involved in several responses, including antipsychotic activity, response to stress and depression, mechanism of sensitization to abused drugs (Ujike et al., 1992; McCracken et al., 1999; Romieu et al., 2000), and marked antiamnesic properties against several models of amnesia (for reviews, see Maurice and Lockhart, 1997; Maurice et al., 1999). The noradrenergic and glutamatergic neurotransmissions play an important role in behavioral despair (Bunney and Davis, 1965; Trullas and Skolnick, 1990). Consequently, the ₁-receptor ligands present effective antidepressant effects in several animal models. The selective ₁-receptor agonists (+)-pentazocine, (+)-SKF-10,047, or SA4503 reduced the immobility time in the forced swimming test (Matsuno et al., 1996) and in the tail suspension test (Ukai et al., 1998). OPC-14523 also showed an antidepressant-like effect in the forced swimming test (Tottori et al., 1997). The selective ₁-receptor antagonist NE-100 reversed the drug-induced reduction of immobility. Igmesine also exhibited a marked antidepressant-like effect in the rat forced swimming test and in the mouse tail suspension test (Kinsora et al., 1998), and has been tested in a clinical trials in humans with promising results (Pande et al., 1998).

The interaction between neuroactive steroids and the ₁-receptor have been uncovered in binding studies (Su et al., 1988; Yamada et al., 1994; Maurice et al., 1996), then reported in physiological studies regarding several neuronal responses (Monnet et al., 1995; Debonnel et al., 1996), with major consequences in several models of amnesia (Maurice et al., 1997, 1998, 1999; Urani et al., 1998). It appeared from these studies that DHEA, pregnenolone, or their sulfate esters behave as ₁-receptor agonists, whereas progesterone behaves as a potent antagonist. Allopregnanolone does not interact with the ₁-receptor. In the forced swimming test, Reddy et al. (1998) showed that the reduction of immobility induced by DHEAS or pregnenolone sulfate was reversed by the ₁-selective antagonist NE-100. Similar results were observed in a conditioned fear stress model (Noda et al., 2000).

In this study, we characterized the antidepressant-like effect of several selective ₁-receptor agonists and the neuroactive steroids that act as ₁-receptor agonists (DHEAS or pregnenolone sulfate) using the forced swimming test in the Swiss mice. We report, through endocrine manipulationsadrenalectomy/castration and inhibition of the 3-hydroxysteroid dehydrogenase that converts pregnenolone into progesterone or of the 5-reductase that metabolizes progesterone into 3-pregnane-3,20-dionethat the endogenous steroidal levels play a major influence on the ₁-receptor availability and behavioral effect. In addition, a biochemical study examined the effect of swimming stress on the ₁-receptor/neurosteroidal systems, through in vivo and in vitro (+)-[³H]SKF-10,047 binding to ₁-sites and measurement of the neurosteroid contents in different brain structures of mice according to the different endocrine manipulations.

	Materials and Methods

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Animals. Male Swiss OF1 mice (Breeding Center of the Faculty of Pharmacy, Montpellier, France), aged 5 to 6 weeks and weighing 30 ± 2 g, were used throughout the study. Animals were housed in groups in plastic cages. They had free access to laboratory chow and water, except during behavioral experiments, and they were kept in a regulated environment (23 ± 1°C, 40-60% humidity) under a 12-h light/dark cycle (light on at 7:00 AM). Experiments were carried out between 9:00 AM and 6:00 PM, in a soundproof and air-regulated experimental room, to which mice were habituated at least 30 min before each experiment. All animal procedures were conducted in strict adherence to the European Community Council Directives of November 24, 1986 (86-609/EEC) and the Decree of October 20, 1987 (87-848).

Drugs. (+)-N-Cyclopropylmethyl-N-methyl-1,4-diphenyl-1-ethyl-but-3-en-1-ylamine hydrochloride (igmesine, CI-1019, JO-1784) was synthesized at Pfizer, Fresnes. Finasteride, desipramine, and progesterone (4-pregnene-3,20-dione) were purchased from Sigma (St. Quentin Fallavier, France). Fluoxetine, pregnenolone sulfate (5-pregnen-3-ol-20-one sulfate), DHEAS (5-androsten-3-ol-17-one sulfate), and (+)-SKF-10,047 were from Sigma/RBI (Natick, MA). 2-(4-Morpholinoethyl)-1-phenylcyclohexane-1-carboxylate hydrochloride (PRE-084) was provided by Dr. D.W. Parish (SRI International, Menlo Park, CA) and N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine (BD1047) by Dr. Wayne D. Bowen (National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health, Bethesda, MD). Trilostane was a generous gift from Dr. G. Margetts (Stegram Pharmaceuticals, Billinghurst, UK). (+)-[³H]SKF-10,047 (1820 GBq/mmol, 37 MBq/ml), [1,2,6,7-³H(N)]progesterone (3589 GBq/mmol, 37 MBq/ml), [7-³H(N)]pregnenolone ([³H]pregnenolone, 777 GBq/mmol, 37 MBq/ml), and [1,2,6,7-³H(N)]dehydroepiandrosterone (2220 GBq/mmol, 37 MBq/ml) and [7-³H(N)]dehydroepiandrosterone sulfate (592 GBq/mmol, 37 MBq/ml) were from PerkinElmer Life Science Products (Boston, MA). The pregnenolone antibody was from AbCys (Paris, France), and progesterone and DHEA antibodies were from Biovalley (Marne-la-Vallée, France). Progesterone and finasteride were suspended in sesame oil; other drugs were solubilized in distilled water or saline solution. Drugs were injected subcutaneously (s.c.) or intraperitoneally (i.p.), in a volume of 100 µl/20 g of body weight.

Forced Swimming Test. Each mouse was placed individually in a glass cylinder (diameter 12 cm, height 24 cm) filled with water at a height of 12 cm. Water temperature was maintained at 22-23°C. The animal was forced to swim for 15 min on the 1st day. Animals were then allowed to return to their home cage. On the 2nd day, each mouse was placed again into the water and forced to swim for 6 min. The session was videotaped and the duration of immobility during the last 5 min was measured. The mouse was considered as immobile when it stopped struggling and moved only to remain floating in the water, keeping its head above water. Drugs were administered 30 min before the session on the 2nd day. Finasteride was administered twice, 14 and 2 h before the session on day 2.

Adrenalectomy/Castration. About 1 week after habituation to the animal facilities, animals were anesthetized with sodium pentobarbital 2%, 100 µl/30 g of body weight i.p. Both adrenal glands were removed, through incisions in the back of the animal, just below the breast ribs. The skin was sutured. Then, both testes were ligatured and cut through an incision in the scrotum. Animals received an injection of gentamicin 10 mg/kg i.p. and recovered within a few hours from surgery. After surgery, drinking tap water was replaced by a saccharose 1%, NaCl 0.9% solution. Animals were used for behavioral experiments 6 days after surgery.

In Vivo (+)-[³H]SKF-10,047 Binding Assays. Mice were injected in a tail vein with 150 kBq of (+)-[³H]SKF-10,047 and sacrificed 30 min later by decapitation. The hippocampi, cortex, and cerebellum were dissected out on ice, homogenized in a 5 mM Tris-HCl buffer, pH 7.4, at 4°C. Two 1-ml aliquots were filtered under vacuum through GF/C filters, presoaked in 0.05% polyethylenimine (Sigma, St. Louis, MO). Total radioactivity was determined by counting 200-µl aliquots of the homogenates. Preliminary experiments showed that the nonspecific binding, defined as the binding levels measured after the preadministration of haloperidol, 2 mg/kg i.p., 10 min before the tracer, represents between 5 to 9% of the total binding (Maurice et al., 1996; Phan et al., 1999). Therefore, results were expressed as total binding and calculated as bound to free radioactivity ratios (Maurice et al., 1996; Phan et al., 1999).

In Vitro (+)-[³H]SKF-10,047 Binding. Control or AdX/CX animals, nonstressed or submitted to the forced swimming test 30 min before, were sacrificed by decapitation. The hippocampi were dissected out at 4°C, pooled, and homogenized in 25 volumes of 50 mM Tris/HCl buffer, pH 7.4, using a Polytron homogenizer for 20 s. The homogenate was centrifuged for 15 min at 45,000g at 4°C. The pellet was resuspended in 5 mM Tris/HCl buffer, pH 7.4. The homogenate was centrifuged again for 15 min at 45,000g at 4°C and the pellet resuspended in the same buffer at a final concentration of 2 mg of protein/ml. Various concentrations of (+)-[³H]SKF-10,047, ranging from 1 nM to 1 µM, were incubated in a total volume of 2 ml of 5 mM Tris/HCl buffer for 60 min at 25°C. The bound radioactivity in 500-µl aliquots was separated by filtration through Whatman GF/C filters presoaked with 0.05% polyethylenimine. The total radioactivity was determined by counting 100-µl aliquots of the homogenates. The nonspecific binding levels were determined using NE-100 (100 µM). The protein concentration in the homogenates was determined using bovine serum albumin (Sigma) as standard (Bradford, 1976).

Extraction and Purification of Neurosteroids. Mice were sacrificed by decapitation, and the brains were quickly removed, dissected, and pooled to obtain approximately 500 mg of tissue. Samples were frozen immediately in dry ice until analysis. The samples were then weighed and homogenized in ice-cold 10 mM phosphate-buffered saline, pH 7.4. Recovery tracers ([³H]progesterone, [³H]pregnenolone, [³H]DHEA, [³H]DHEAS, 50 Bq each) were added. Then, 10 ml of ethyl acetate/isooctane, 1:1 v/v, was added and the tubes were vigorously stirred for 8 min. After centrifugation at 4,000g for 5 min, the organic phase was removed and the extraction step was repeated twice. This organic phase was then defatted with a MeOH 90%/isooctane separation. The aqueous extracts containing unconjugated steroids were further purified by reverse-phase chromatography on Amprep C₁₈ cartridges (Amersham, Les Ulis, France). The isooctane phases containing lipoidal derivatives were thrown away. Sulfate esters were solvolyzed. The aqueous phase from the first separation was brought to pH 1.0 with a few drops of sulfuric acid and to a NaCl concentration of 20% by adding 2:1 v/v of a 30% NaCl solution. Extraction with ethyl acetate was again performed as described above, and this extract, which contained steroid sulfates, was solvolyzed at 37°C for 16 h. Ethyl acetate extracts were washed once with 1 N NaOH (0.25 volume) and twice with water (0.25 volume). The extracts were taken to dryness.

Extraction and Purification of Plasma Steroids. Mice were anesthetized with pentobarbital 2%, and blood was collected through transcardiac punction in heparinized tubes. Samples were centrifuged 10 min at 4,000g and pooled to obtain 2 ml of plasma. Then, 3 ml of phosphate-buffered saline were added, and extraction/purification of steroids was performed as described.

Radioimmunoassays. After separation, each steroid was quantified by radioimmunoassay using specific antibodies presenting minimal cross-reactions. Measurements were performed in triplicate of four dilutions of each purified sample. Results are expressed as nanograms per milliliter of plasma or nanograms per gram of tissue.

Statistical Analysis. Results are expressed as means ± S.E.M. Data were analyzed using the Dunnett's multiple comparisons test after analysis of variance (F values). For the neurosteroid measurement, statistical comparison of the data was made using the Welch's test. The criteria for statistical significance were P < 0.05, P < 0.01, and P < 0.001.

	Results

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Effects of ₁-Receptor Agonists on the Immobility Time in the Forced Swimming Test. When submitted to forced swimming, Swiss mice rapidly developed a marked behavioral despair, showing an immobility time of 180 ± 4 s (n = 18) on the 1st day (recorded between the 2nd and 6th minute of swimming), which significantly increased up to 227 ± 5 s on the 2nd day (t = 7.92, P < 0.01, paired t test). Consequently, drugs were administered before the session on day 2. The selective ₁-receptor agonist igmesine significantly shortened the immobility time at the dose of 60 mg/kg i.p. [F_(4,75) = 4.43, P < 0.01; Fig. 1A]. Similarly, the reference ₁-receptor agonist (+)-SKF-10,047 decreased the immobility time, at the dose of 30 mg/kg [F_(4,40) = 7.72, P < 0.01; Fig. 1B]. However, the other selective ₁-receptor agonist PRE-084 failed to affect the immobility time in the 10 to 60 mg/kg dose range [F_(4,47) = 0.75, P > 0.05; Fig. 1C]. Lower doses were also tested (1-5 mg/kg) that appeared ineffective. The selective ₁-receptor antagonist BD1047, tested in the 0.3 to 3 mg/kg i.p. dose range, did not affect the immobility time by itself [F_(3,32) = 1.70, P > 0.05; Fig. 1D]. However, preadministration of BD1047, at 3 mg/kg, completely antagonized the reduction of immobility time induced by igmesine [F_(4,49) = 33.02, P < 0.001; Fig. 1E], confirming that the drug acted through the involvement of the ₁-receptor.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Antidepressant-like effect of 1-receptor ligands in the forced swimming test in Swiss mice: dose-response effect of igmesine (A), (+)-SKF 10,047 (B), PRE-084 (C), BD1047 (D), and antagonism by BD1047 of the igmesine-induced effect (E). Drugs were injected i.p. 30 min before the session on day 2. BD1047 was administered i.p. 15 min before igmesine, which was given 30 min before the session on day 2. The duration of immobility was recorded for the last 5 min over a 6-min session. Values are expressed as mean ± S.E.M. of the number of animals indicated inside each column. *P < 0.05, **P < 0.01 versus the vehicle-treated group (Veh); ##P < 0.01 versus the igmesine-treated group (Dunnett's test).

Effects of Neuroactive Steroids on the Immobility Time in the Forced Swimming Test. DHEAS, administered in the 5 to 60 mg/kg dose range, slightly but significantly shortened the immobility time, at the 10 mg/kg dose [F_(5,89) = 2.80, P < 0.05; Fig. 2A]. At lower or higher dosages, the steroid was devoid of effect. Pregnenolone sulfate failed to affect the immobility time in the 5 to 40 mg/kg dose range [F_(4,74) = 1.74, P > 0.05; Fig. 2B]. Progesterone did not affect the immobility time by itself in the 5 to 60 mg/kg dose range [F_(5,68) = 0.21, P > 0.05; Fig. 2C]. However, the preadministration of progesterone (20-60 mg/kg) before DHEAS (10 mg/kg) led to a significant antagonism of its effect, as shown in Fig. 2D [F_(3,55) = 6.46, P < 0.001].

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Antidepressant-like effect of neuroactive steroids in the forced swimming test in Swiss mice: dose-response effect of DHEAS (A), pregnenolone sulfate (B), progesterone (C), and antagonism by progesterone of the DHEAS-induced effect (D). The steroids were injected i.p. 30 min before the session on day 2. Progesterone was administered i.p. 15 min before DHEAS, which was given 30 min before the test. Values are expressed as mean ± S.E.M. of the number of animals indicated inside each column. *P < 0.05, **P < 0.01 versus the vehicle-treated group (Veh); ##P < 0.01 versus the igmesine-treated group (Dunnett's test).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Antagonism of the antidepressant-like effects of igmesine by progesterone (PROG) (A) and of DHEAS by BD1047 (B) in Swiss mice. Progesterone or BD1047 was administered i.p. 15 min before igmesine or DHEAS, respectively, which was given 30 min before the test. Values are expressed as mean ± S.E.M. of the number of animals indicated inside each column. *P < 0.05, **P < 0.01 versus the vehicle-treated group (Veh); ##P < 0.01 versus the igmesine- or DHEAS-treated group (Dunnett's test).

Effects of ₁-Receptor Ligands on the Immobility Time in the Forced Swimming Test in AdX/CX Animals. AdX/CX animals showed an immobility time of 178 ± 12 s (n = 12) on the 1st day, which significantly increased up to 242 ± 5 s on the 2nd day (t = 4.26, P < 0.01, paired t test). These values did not differ from the values measured in control animals (P > 0.05 on each day). Igmesine shortened significantly the immobility time in AdX/CX animals, in a dose-dependent manner at 20 and 60 mg/kg [F_(5,49) = 18.70, P < 0.001; Fig. 4A], thus in a more effective manner as compared with nonoperated animals (Fig. 1A). The finasteride pretreatment, which led to an increase in the endogenous levels of progesterone, did not affect the immobility time exhibited by vehicle-treated AdX/CX mice, but significantly altered the diminution induced by igmesine, at 60 mg/kg (Fig. 4A). PRE-084 allowed a significant and dose-dependent decrease of immobility time in AdX/CX animals, at 20 and 60 mg/kg [F_(5,58) = 6.16, P < 0.001; Fig. 4B]. In the same dose range, the compound was without effect in control animals (Fig. 1C). The finasteride pretreatment significantly affected the effect-induced PRE-084, 60 mg/kg, in AdX/CX animals (Fig. 4B).

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Antidepressant-like effect of igmesine (A) and PRE-084 (B) in the forced swimming test in AdX/CX Swiss mice. Surgery was performed 7 days before the test. Half of experimental groups were treated with finasteride (25 mg/kg s.c.) 14 and 2 h before the session on day 2. The 1-ligands were injected i.p. 30 min before the session. Values are expressed as mean ± S.E.M. of the number of animals indicated inside each column. **P < 0.01 versus the vehicle-treated (Veh) group; ##P < 0.01 versus the igmesine- or PRE-084-treated group (Dunnett's test).

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Antidepressant-like effect of desipramine (A, B) and fluoxetine (C, D) in the forced swimming test in control (A, C) and AdX/CX (B, D) Swiss mice. In (B, D), surgery was performed 7 days before the test. Drugs were injected i.p. 30 min before the session on day 2. Values are expressed as mean ± S.E.M. of the number of animals indicated inside each column. *P < 0.05, **P < 0.01 versus the vehicle-treated (Veh) group (Dunnett's test).

Effects of Neuroactive Steroids on the Immobility Time in the Forced Swimming Test in AdX/CX Animals. In AdX/CX animals, both DHEAS and pregnenolone sulfate significantly shortened the immobility time, as shown in Fig. 6. The DHEAS treatment led to significant diminution of the immobility time at all the doses examined, within the 5 to 60 mg/kg dose range [F_(5,56) = 10.86, P < 0.001; Fig. 6A], thus in a more efficient manner as compared with nonoperated animals (Fig. 2A). The pregnenolone sulfate treatment led to significant reductions of the immobility time at doses of 20 and 40 mg/kg [F_(4,30) = 3.24, P < 0.05; Fig. 6B]. In the same dose range, the steroid was without effect in control animals (Fig. 2B).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Antidepressant-like effect of DHEAS (A) and pregnenolone sulfate (B) in the forced swimming test and antagonism of the antidepressant-like effect of igmesine (C) or DHEAS (D) by BD1047 in AdX/CX Swiss mice. Surgery was performed 7 days before the test. Steroids or igmesine were injected i.p. 30 min before the session. BD1047 was administered i.p. 15 min before igmesine or DHEAS. Values are expressed as mean ± S.E.M. of the number of animals indicated inside each column. **P < 0.05, **P < 0.01 versus the vehicle-treated group (Veh); ##P < 0.01 versus the igmesine- or DHEAS-treated group (Dunnett's test).

In Vivo (+)-[³H]SKF-10,047 Binding Measures after Swimming Stress. The availability of the ₁-receptor after stress and following different endocrine manipulations was examined in brain structures selected for: i) their involvement in the response to stress, ii) their role in mediating the cognitive effects of ₁-agonists, and iii) the possibility to measure the neurosteroid levels using the extraction/purification/radioimmunoassay technique, i.e., the hippocampi and cortex, plus a control structure, the cerebellum. In control animals submitted to a swimming stress (Fig. 7), a significant reduction of the in vivo (+)-[³H]SKF-10,047 binding level was observed in the hippocampus [F_(4,22) = 5.74, P < 0.001; Fig. 7A]. The inhibition was observed immediately after the stress and at the 30-min timepoint, when a 27% reduction was measured. No change was observed in the cortex [F_(4,22) = 1.01, P > 0.05; Fig. 7B] and cerebellum [F_(4,22) = 0.10, P > 0.05; Fig. 7C]. The immobility duration was checked and did not differ among experimental groups [F_(3,19) = 1.41, P > 0.05; Fig. 7D]. In AdX/CX animals submitted to a swimming stress, the in vivo (+)-[³H]SKF-10,047 binding level significantly decreased in the hippocampus [F_(4,23) = 5.62, P < 0.001; Fig. 8A]. The inhibition was observed immediately after stress and remained significant until the 60-min timepoint. At 30 min after swimming stress, a 48% reduction was measured. No change was observed in the cortex [F_(4,23) = 1.08, P > 0.05; Fig. 8B] and cerebellum [F_(4,23) = 0.44, P > 0.05; Fig. 8C]. In addition, the immobility duration did not differ among experimental groups, in the 231 to 266 s range [F_(3,19) = 2.30, P > 0.05; data not shown]. Finally, AdX/CX animals were treated twice a day with trilostane (10 mg/kg s.c.) and the in vivo (+)-[³H]SKF-10,047 binding levels measured after the swimming stress. In these conditions, no significant reduction could be observed in the hippocampus [F_(4,29) = 0.36, P > 0.05; Fig. 8D], as well as in the cortex [F_(4,29) = 1.09, P > 0.05; Fig. 8E] or cerebellum [F_(4,28) = 0.62, P > 0.05; Fig. 8F]. Here, again, the immobility duration did not differ among experimental groups, in the 229 to 246 s range [F_(3,19) = 1.89, P > 0.05; data not shown].

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Time course of the in vivo (+)-[3H]SKF-10,047 binding levels after a swimming stress in Swiss mice. Binding levels in the hippocampus (A), cortex (B), cerebellum (C), and immobility time of each experimental group (D). Animals were submitted to forced swimming for 15 min on day 1 and 6 min on day 2, immobility being recorded during the last 5 min. The in vivo binding assays were performed at varying intervals after swimming, the radioligand being injected 30 min before decapitation. Open circles, nonstressed control animals (n = 3). The number of animals per group is indicated within the column in (D). *P < 0.05, **P < 0.01 versus nonstressed controls (Dunnett's test).

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Time course of the in vivo (+)-[3H]SKF-10,047 binding levels after a swimming stress in the hippocampus (A), cortex (B), and cerebellum (C) of AdX/CX mice, and in the hippocampus (D), cortex (E), and cerebellum (F) of AdX/CX mice treated with trilostane. Animals were submitted to forced swimming for 15 min on day 1 and 6 min on day 2, immobility being recorded for the last 5 min. The in vivo binding assays were performed at varying intervals after swimming, the radioligand being injected 30 min before decapitation. Open circles, nonstressed animals. The number of animals per group was n = 5 to 9. *P < 0.05, **P < 0.01 versus nonstressed controls (Dunnett's test).

In Vitro (+)-[³H]SKF-10,047 Binding Parameters after Swimming Stress. Since marked decreases of the in vivo (+)-[³H]SKF-10,047 binding levels were observed in the hippocampus of control or AdX/CX animals, the binding parameters (K_d, B_max) were determined in saturation experiments (Table 1). Hippocampal membranes were prepared from control and/or AdX/CX animals and from animals submitted to a swimming stress 30 min before. The Scatchard analyses resulted in a single population of site with a dissociation constant, K_d value, about 6 nM and a density of sites, B_max, about 60 fmol/mg of protein in control animals (Table 1). The stress and/or surgery failed to affect these parameters, indicating that the binding parameters of the ₁-receptor are unaffected following such endocrine manipulations (Table 1).

Steroid Contents. Plasma levels of circulating progesterone were first measured to validate the endocrine manipulations (Table 2). Adrenalectomy/castration drastically decreased the progesterone levels almost under the detection limits for both nonstressed and stressed animals. In addition, the swimming stress resulted in a 2-fold increase of the progesterone levels, although not significant (Table 2).

View larger version (30K): [in this window] [in a new window]   Fig. 9.   Brain contents in progesterone in the hippocampus (A), cortex (B), cerebellum (C), and whole brain (D) of Swiss mice. Progesterone levels were measured in intact, AdX/CX mice, or AdX/CX mice treated for 7 days with trilostane, and levels in nonstressed () or stressed () mice are compared. Stressed animals were sacrificed 30 min after swimming. The number of samples was n = 4 to 8. *P < 0.05, **P < 0.01 versus nonstressed controls; #P 0.05, ##P < 0.01 versus the corresponding AdX/CX group (Welch's test).

	Discussion

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This study characterized the potential antidepressant-like effect exerted by selective ₁-receptor agonists, which was previously suggested in several rodent models of behavioral despair (Matsuno et al., 1996; Tottori et al., 1997; Kinsora et al., 1998; Ukai et al., 1998). We report here that the prototypic ₁-receptor agonist, (+)-SKF-10,047, as well as a more selective compound like igmesine, significantly reduced the immobility time exhibited by Swiss mice in the forced swimming test. The extent of the effect markedly varied among compounds and depending on experimental conditions. In particular, PRE-084 failed to show any effect in control animals. The selective ₁-receptor antagonist BD1047 antagonized the igmesine-mediated effect, clearly demonstrating the involvement of the ₁-receptor. Matsuno et al. (1996) previously reported that, in the same forced swimming test, several ₁-receptor agonists, including SA4503, igmesine, and 1,3-di-o-tolylguanidine, presented potent antidepressant-like effect. Our results are in full agreement, except as concerns the active doses of the compounds. Such differences may however be attributable to the different mouse species used in each study: ddY for Matsuno et al. (1996) and Swiss for the present study. Interestingly, the ₁-receptor ligands appeared almost as effective as antidepressant drugs used in human clinical studies, such as fluoxetine or desipramine, with a only a mild shift in the active dose. Such observation confirmed the therapeutical interest for the use of selective ₁-receptor agonists in depression treatment, particularly considering their lack of deleterious side effects (Matsuno and Mita, 1998).

The main aspect of this study was to investigate the interaction between the ₁-receptor and the neuroactive steroids in such model of depression. This interaction was first described using in vitro and in vivo binding measures of [³H](+)-SKF-10,047 to the ₁-site (Su et al., 1988; Maurice et al., 1996). Progesterone appeared as the most potent inhibitor of the in vitro [³H](+)-SKF-10,047 binding in the rat brain, with a K_i value around 300 nM (Su et al., 1988). In vivo, progesterone, pregnenolone sulfate, and in a lesser extent DHEAS, decreased the [³H](+)-SKF-10,047 binding levels in the mouse brain, demonstrating the validity of this interaction in vivo (Maurice et al., 1996). Furthermore, DHEAS or pregnenolone sulfate behaved as agonists and progesterone as an antagonist in several tests assessing the ₁-receptor pharmacology in vitro and in vivo (Monnet et al., 1995; Debonnel et al., 1996; Hayashi et al., 2000). In particular, the effect of DHEAS, the potentiation of N-methyl-D-aspartate-evoked responses, was blocked by selective ₁-receptor antagonists and progesterone blocked the ₁-receptor agonist-mediated effect, as well as the steroidal effect. The effect of pregnenolone sulfate appeared much more tenuous, the steroid behaving as an agonist or inverse agonist in vitro and being ineffective in vivo (Monnet et al., 1995; Debonnel et al., 1996; Hayashi et al., 2000). A similar crossed pharmacology between the ₁-receptor ligands and the neuroactive steroids was observed at the behavioral level in several amnesia models. Selective ₁-receptor agonists are potent antiamnesic drugs, an effect shared by pregnenolone sulfate or DHEAS (Maurice et al., 1997, 1998; Maurice and Privat, 1997; Urani et al., 1998). Progesterone acted similarly as selective ₁-receptor antagonists in these different tests.

In depression models, these neuroactive steroids were previously reported to have some effect. First, DHEA alleviated behavioral despair in high-anxiety rats (Prasad et al., 1997). Second, DHEAS and pregnenolone sulfate reduced the immobility time in mice submitted to the forced swimming test. We showed here that, in control Swiss mice, DHEAS slightly but significantly reduced the immobility time, and pregnenolone sulfate was inactive. Progesterone behaved as an antagonist and blocked the DHEAS effect. The crossed pharmacology between neuroactive steroids and ₁-ligands could be observed in this depression model, since progesterone blocked the effect of ₁-agonists while the ₁-antagonist BD1047 blocked the DHEAS effect. Moreover, progesterone affected the in vivo binding in the mouse hippocampus and cortex at doses higher than 10 mg/kg, doses relevant to the antagonist effect described in the present study. It appears therefore that the endogenous (neuro)steroidal systems interfere with the antidepressant-like effects mediated through the ₁-receptor. It was however observed that in control Swiss mice, the active steroid, DHEAS, presented only a limited and bell-shaped effect in the forced swimming test. Reddy et al. (1998) reported a similar narrow dose range and bell-shaped effect for both DHEAS and pregnenolone sulfate, the effectiveness of steroids being observed at 5 mg/kg. In the present study, the effective dosage for DHEAS was limited to 10 mg/kg.

To further investigate the relationship between the steroidal system and the ₁-receptor in the forced swimming test, endocrine manipulations were carried out. Animals were adrenalectomized and castrated (AdX/CX) to remove the peripheral sources of steroids. Indeed, peripheral steroids do cross the blood-brain barrier and contaminate the brain levels of neurosteroids. Animals were then treated with finasteride, an inhibitor of the 5-reductase enzyme that convert progesterone to 5-pregnane-3,20-dione. This treatment led to an accumulation of neurosteroidal progesterone in the brain of AdX/CX animals (A. Urani, V. L. Phan, P. Romieu, and T. Maurice, manuscript in preparation). Furthermore, we previously reported that, in Swiss AdX/CX mice, these endocrine manipulations have major consequences on: i) the in vivo [³H](+)-SKF-10,047 binding levels in the hippocampus and cortex and ii) the extent of the antiamnesic effects of PRE-084 against the dizocilpine-induced learning deficits (Phan et al., 1999). In this study, we characterized the ₁-receptor-mediated antidepressant-like effect in AdX/CX animals. The fact that adrenal glands were removed is of importance using a behavioral test involving a response to stress, and it is likely that AdX/CX mice did not respond similarly to forced swimming as do control animals. However, several observations led us to consider that they performed the test in a valuable manner. In particular, the immobility duration measured for AdX/CX animals did not differ from the values measured in control animals or sham-operated ones (data not shown), during the pretest on day 1 or during the test on day 2. In addition, the maximal effects of the ₁-receptor agonists were fully antagonized by BD1047 (see data herein). The effect of igmesine was enhanced in AdX/CX animals, as compared with nonoperated mice. Igmesine appeared active at 10 mg/kg, whereas in intact animals the effect appeared only at 60 mg/kg. Moreover, the surgery revealed an antidepressant-like effect for PRE-084. The compound, which had no effect in intact animals, presented a significant effect at doses higher than 20 mg/kg in AdX/CX animals. Interestingly, however, it remained less effective than igmesine in reducing the immobility time at similar dosage, confirming that the compound may present a lower intrinsic efficacy as antidepressant, compared with igmesine. The results in AdX/CX mice suggested that circulating steroids exert a tonic modulatory effect on the ₁-receptor-mediated antidepressant-like effect. The potent role of endogenous antagonist exerted by progesterone is further evidenced by the observation that the treatment with finasteride, i.e., accumulation of the neurosteroidal progesterone in the brain, blocked the ₁-receptor-mediated effect.

Interestingly, the effect of the antidepressant drugs fluoxetine and desipramine was not affected by i) progesterone preadministratrion and ii) removal of circulating steroids, clearly indicating that the mechanism of the ₁-receptor-mediated antidepressant-like effect is different from the effect induced by classical antidepressants and does not involve an indirect inhibition of monoamine transporters. It has been suggested that fluoxetine may also act by correcting the imbalance between several neurosteroids, as observed during major depression (Romeo et al., 1998). The steroids concerned are 5-pregnane-3-ol-20-one, 5-pregnane-3-ol-20-one, and 5-pregnane-3-ol-20-one, which are potent modulators of the GABA_A receptor and do not interact with ₁-receptor. The relative importance of this alternative mechanism as compared with the direct effect described in the present paper remains to be further investigated.

In AdX/CX animals, the effect of the neuroactive steroids was also enhanced. The extent of the DHEAS effect was increased, and the steroid was effective at all the doses tested, although it reduced immobility only at 10 mg/kg in intact animals. Pregnenolone sulfate, which was ineffective in intact animals, had an antidepressant-like effect in AdX/CX animals, similar to what was observed with PRE-084. Moreover, in AdX/CX animals, the effect of DHEAS, like the effect of igmesine, was completely blocked by BD1047. Taken together, these results suggest that the limited effect induced by DHEAS in control animals and the lack of effect of pregnenolone sulfate are due to the impeding effect of endogenous progesterone. It also confirmed that the mechanism of the antidepressant-like actions of neuroactive steroids and ₁-receptor agonists are closely related.

The second part of the present study examined the parallel changes between the in vivo binding levels at the ₁-receptor, labeled using (+)-[³H]SKF-10,047, and the contents in several neuroactive steroids, in the brain of the Swiss mouse submitted to an acute stress induced by forced swimming. In the hippocampus, the in vivo binding levels of (+)-[³H]SKF-10,047 decreased immediately after stress (27%), and this effect persisted significantly until 30 min after the stress. Such an effect was not observed in the other structures examined, namely the cortex and cerebellum. This effect was enhanced in AdX/CX animals, deprived of circulating steroids: 48% inhibition and longer in duration, remaining significant until 60 min after stress. However, it was completely blocked in AdX/CX animals treated with trilostane, a 3-hydroxysteroid dehydrogenase inhibitor that blocks the formation of progesterone.

In nonstressed animals, the in vivo (+)-[³H]SKF-10,047 binding levels were higher in AdX/CX animals compared with controls and furthermore in trilostane-treated animals, confirming previous observations (Phan et al., 1999). The higher levels of binding in AdX/CX animals is likely to reflect not only the decreased levels of circulating progesterone in plasma that can almost be considered as close to the detection limits after surgery (Table 2), but also the deprivation of other steroids bearing some affinity for the ₁-sites, including, but not limited to, testosterone or desoxycorticosterone (Su et al., 1988). The hippocampal levels of progesterone were unchanged after surgery, but appeared highly significantly decreased after the trilostane treatment (Fig. 4), confirming that the levels of in vivo (+)-[³H]SKF-10,047 binding in basal conditions are negatively correlated to the progesterone contents in the brain.

The transient decrease of in vivo (+)-[³H]SKF-10,047 binding levels in the hippocampus after acute swimming stress may be related to either particular changes in the binding parameters of the ₁-receptor, including reduction in apparent affinity or modification of the association or dissociation rates, or the release of endogenous neurotransmitter or hormones, including the putative but as yet unidentified endogenous ligand "sigmaphin" (Su et al., 1986). Modifications of the binding parameters of the ₁-receptor were assessed using saturation experiments in vitro. It was clearly observed that neither the surgery nor the acute stress affected significantly the apparent affinity and the number of sites under our experimental conditions. It appeared thus that the hypothesis of a transient release of endogenous modulator, shortly after stress, might be relevant. The neurosteroidal progesterone appeared as the main candidate according to the following observations. i) Increases in progesterone appeared the most pronounced in the hippocampus, compared with the other structures examined and as observed for the changes in the in vivo (+)-[³H]SKF-10,047 binding. ii) Variations in the hippocampal progesterone content correlated closely to the inhibition of binding. iii) The changes, induced after the endocrine manipulations and acute stress, of the brain contents in the other steroids appeared unrelated to the selective inhibition of hippocampal in vivo binding to the ₁-sites. Indeed, no difference was found between intact and AdX/CX mice, in accordance with previous reports showing that adrenalectomy/castration does not affect neurosteroid content (Corpéchot et al., 1981, 1983; Purdy et al., 1991). Stress induced an increase in pregnenolone level in intact animals as previously described (Barbaccia et al., 1996). Stress also induced an increase in DHEAS content in the cortex and whole brain of intact animals and in the cortex of AdX/CX animals. iv) Progesterone plays a particular role as a neuromodulatory hormone: first, as precursor of allopregnanolone and other related stress-induced steroids and second, as the steroid presenting the highest affinity for the ₁-receptor (Su et al., 1988; Maurice et al., 1996) and behaving as a potent antagonist (Monnet et al., 1995; Bergeron et al., 1996; Maurice and Privat, 1997).

It can thus be proposed that progesterone is released during stress, not only in periphery, but also centrally; this release exerts a blockade of the ₁-receptor. The release of progesterone in response to stress appeared however as an important physiological response (Purdy et al., 1991; Duncan et al., 1998). Interestingly, it was observed that when peripheral progesterone is lacking, in AdX/CX animals, the brain is able to partly compensate by increasing the release of progesterone from neurosteroidal origin (from +80% to +150%). Selective ₁-receptor agonists, when injected systemically, would compete with the high levels of progesterone to exert their pharmacological effect as antidepressants.

Finally, the biochemical data confirmed the behavioral observations. The antidepressant-like effects of the ₁-agonists from synthetic or steroidal origin, namely igmesine, PRE-084, DHEAS, or pregnenolone sulfate, in the forced swimming test was highly dependent on the endogenous progesterone levels following different endocrine manipulations. The effect of igmesine or DHEAS observed in intact animals was enhanced in AdX/CX animals. This result can be related to the observation that the in vivo (+)-[³H]SKF-10,047 binding level is increased, compared with intact animals. Furthermore, accumulation of progesterone using a treatment with finasteride, a 5-reductase inhibitor that blocks the metabolism of progesterone, attenuated the efficacy of the ₁-agonists.

In conclusion, these results are in accordance with clinical studies showing that neurosteroids, and DHEA in particular, may be potent antidepressants (Wolkowitz et al., 1997, 1999). Moreover, we show here that the antidepressant-like effect of neuroactive steroids is mediated by a direct interaction with the ₁-receptor. Our results also demonstrate that the efficacy as antidepressant critically depends on the hormonal status of the animal. An acute stress induces the release of progesterone from the peripheral and central sources, which in turn interacts transiently with the ₁-receptor. This negative regulation, since progesterone behaves as a potent ₁-antagonist, must be overridden by the selective synthetic ₁-agonists to exert their antidepressant-like effect. These observations are of clinical importance, suggesting that the (neuro)steroidal levels of patients are to be taken in account in case of antidepressant therapy involving ₁-agonist. It is almost impossible at present to assess the levels of neurosteroids in vivo in patients. However, depressed patients with decreasing levels of neurosteroids, such as in the elderly, may be particularly sensitive to such therapy.