Journal of Neuroscience Research 46:734-743 (1996)

Rapid Communication Modulation by Neurosteroids of the In Vivo ( +)-[3H]SKF-10,047 Binding to a, Receptors in the Mouse Forebrain Tangui Maurice, Franqois J. Roman, and Alain Privat INSERM U. 336, DCveloppement, Plasticit6 et Vieillissement du Systkme Nerveux, Ecole Nationale Sup6rieure de Chimie, Montpellier (T.M., A.P.), and DCpartement de Biochimie, Institut de Recherche Jouveinal, Fresnes (F.J.R.), France

Recent reports suggest an interaction between neuro- (active)steroids and sigma, (a,) receptors, affecting biochemical parameters as well as physiological re- sponses mediated by u1 ligands in the rodent brain. In this study, we examined the modulation by neuro- steroids of the haloperidol-sensitive in vivo (+)- [3H]SKF'-10,047 binding to u, sites in the mouse hip- pocampus and cortex. Progesterone (PROG; 2 4 0 mglkg), pregnenolone sulfate (PREGS; 10-40 mg/ kg), and dehydroepiandrosterone sulfate (DHEAS; 1 0 4 0 mg/kg) were administered systemically 10 min before the radioactive tracer. The total amount of ( +)-[3H]SKF-10,047 bound in each structure was sig- nificantly affected by PROG and PREGS only at the highest dose tested and was unaffected by DHEAS. However, bound to free (BIF) radioactivity ratios were highly significantly decreased by 3040% in each structure by PROG and PREGS. DHEAS, at 40 mglkg, induced a significant 20% decrease in the hip- pocampus. Furthermore, the in vivo ( + )-[3H]SKF- 10,047 binding parameters were diminished in preg- nant female mice compared to non-pregnant or male mice. These results confirm the in vitro binding re- sults, bring a direct in vivo demonstration of the in- teraction between neurosteroids and u, receptors, and show that physiologic modulations of the steroi- dal concentrations affect the u1 systems. 0 1996 Wiley-Liss, Inc.

Key words: sigma, receptors, neuroactive steroids, in vivo binding, mouse

INTRODUCTION Neuro(active)steroids, such as progesterone

(PROG) , pregnenolone (PREG) , dehydroepiandroster- one (DHEA), and their respective sulfate esters (PREGS,

DHEAS), are involved in regulating the balance between excitation and inhibition in the central nervous system (CNS) (Wu et al., 1991; Paul and Purdy, 1992). They affect the activity of several neurotransmitter systems. In particular, they act as negative allosteric modulators of the GABA, receptor on the barbiturate-mediated en- hancement of benzodiazepine binding (Majewska and Schwartz, 1987; Majewska et al., 1988, 1990). Neuro- steroids also potentiate several responses mediated by the N-methyl-D-aspartate (NMDA) type of glutamatergic re- ceptors. They enhance the NMDA-gated currents in spi- nal cord neurons, increase the convulsant potency of NMDA in mice, modulate the excitatory amino acid- induced responses of cerebellar Purkinje cells, modulate the NMDA-mediated increases in intracellular Ca2 + in cultured rat hippocampal neurons, potentiate the NMDA- evoked [3H]norepinephrine release from rat hippocampal slices, and antagonize the learning impairments induced in mice by competitive and non-competitive NMDA re- ceptor antagonists (Smith, 1991; Wu et al., 1991; Irwin et al., 1992; Maione et al., 1992; Bowlby, 1993; Irwin et al., 1994; Mathis et al., 1994; Cheney et al., 1995; Fa- hey et al., 1995; Monnet et al., 1995; Maurice et al., 1996a). The exact mechanisms of these neuromodulatory steroidal effects induced by neurosteroids are still con- troversial, but appeared to be distinct from their genomic effects.

On the other hand, several steroids, including PROG, PREG, PREGS, testosterone, and 17P-estra- diol, have been shown to significantly inhibit the in vitro binding of the sigma (u) site radioligands (+>- [3H] SKF- 10,047, [3H]dextr~methorphan, ( + )- [3H] 3 -

Received September 4, 1996; accepted September 21, 1996.

Address reprint requests to Dr. Tangui Maurice, INSERM U. 336, ENSCM, 8 rue de 1'Ecole Normale, 34053 Montpellier Cedex 1, France.

0 1996 Wiley-Liss, Inc.

Neurosteroids and In Vivo Binding to ul Sites 735

Male and non-pregnant females were housed in groups of 10. Pregnant female mice were housed individually. An- imals were housed in plastic cages, with free access to standard laboratory food and water, and kept in a regu- lated environment (23 * 1°C) under a 12-hr light/dark cycle (light on at 7:OO A.M.), with tap water and labora- tory food ad libitum.

Drugs ( +)-[3H]SKF-10,047 (54 Ci/mmol, 1 mCi/mmol)

was from the Commissariat a 1'Energie Atomique (Saclay, France). Haloperidol (Haldol@) was from Jans- sen (Boulogne-Billancourt, France). PROG (Cpregnene- 3,20-dione), PREGS (5-pregnen-3P-ol-20-one sulfate), and DHEAS (5-androsten-3P-ol-17-one sulfate) were from Sigma (France). PREGS and DHEAS were solubi- lized in dimethylsulfoxide (Sigma). PROG was sus- pended in pure sesame oil (Sigma). Other compounds and the tracer were solubilized in saline solution. Neu- rosteroids were injected subcutaneously (SC) and halo- peridol intraperitoneally (IP) in a volume of 100 p1/20 g of body weight.

In Vitro ( +)-[3H]SKF-10,047 Binding Assays in Rat Brain Homogenates

The membrane preparation was performed accord- ing to Largent et al. (1986). Animals were killed by decapitation, and whole brains were homogenized in 30 volumes (v/w) ice-cold 50 mM Tris-HC1 pH 7.4 buffer, with a polytron (Kinematica). The homogenates were centrifuged at 20,OOOg for 10 min at 4°C. Pellets were resuspended in 30 volumes of ice-cold buffer and incu- bated for 45 min at 37°C. After a second similar centrif- ugation, the pellets were resuspended in 10 volumes of ice-cold buffer and aliquots stored at -80°C until use. Frozen membrane aliquots were thawed and centrifuged at 20,OOOg for 10 min at 4"C, and pellets were resus- pended in 10 volumes of 5 mM Tris-HC1 pH 7.4 buffer. Incubations were carried out in a volume of 250 pl with 10 nM of ( +)-[3H]SKF-10,047, the competing ligand, and 1.5 mg of proteidml. After 45 min incubation at 25"C, the aliquots were filtered under vacuum through GF/B filters (Whatman, Clifton, NJ), presoaked in 0.05% polyethyleneimine (Aldrich, Milwaukee, WI). Non-specific binding was determined in the presence of 10 pM haloperidol. Radioactivity was measured with 5 ml of Scintillator 299 (Packard, Meriden, CT), by liquid scintillation spectrophotometry (Packard). Protein con- centration was determined by the method of Lowry et al. (1951).

In Vivo ( +)-[3H]SKF-10,047 Binding Assays in Mouse Brain

In vivo ( + )-[3H]SKF-10,047 binding assays were performed as previously described (Bouchard et al.,

PPP, and [3H]haloperidol from rat brain, splenocytes plasma membranes, and liver microsomes (Su et al., 1988; McCann and Su, 1991; Ross, 1991; Klein and Musacchio, 1994; Yamada et al., 1994). This interaction with the u systems could constitute a possible mecha- nism for the steroidal non-genomic effects. Although ini- tially postulated, u sites differ from opioid as well as NMDA receptor-associated phencyclidine sites (Quirion et al., 1992). The unique ligand specificity and autora- diographic distribution of u sites in both the CNS and peripheral tissues suggested that they belong to a distinct receptor family (Walker et al., 1990; Su, 1991). Binding strategies and in vivo and in vitro bioassays provided evidence for the existence of at least of two subtypes of u sites, denoted u1 and u2 (Quirion et al., 1992). The u1 site was recently purified and the cDNA was cloned (Hanner et al., 1996). The amino acid sequence was structurally unrelated to known mammalian proteins, but shared homology with fungal sterol C,-C, isomerase, which seemed in agreement with a putative steroidda, ligands interaction (Hanner et al., 1996). Furthermore, one of the major roles of u1 receptor agonists is the potentiation of several NMDA-mediated responses, such as the NMDA-induced electric activity of rat dorsal hip- pocampal CA, pyramidal neurons, or the NMDA- evoked [3H]norepinephrine release from rat hippocampal slices (Monnet et al., 1990; Roman et al., 1991; Monnet et al., 1992). A crossed pharmacology between the ef- fects of u, ligands and neurosteroids was recently de- scribed on these responses, DHEAS and/or PREGS be- having as agonists and PROG as an antagonist (Monnet et al., 1995; Bergeron et al., 1996). We also recently reported that DHEAS attenuated the learning impairment induced in mice by the non-competitive NMDA antago- nist dizocilpine in a similar manner as do selective u1 ligands, its effect being blocked by the non-selective u1 antagonists haloperidol or BMY-14,802 (Maurice et al., 1996a). Accordingly, we decided to examine further and more directly the possible interaction between neuroster- oids and u1 receptors in the mouse hippocampal forma- tion and cortex by using the in vivo binding assay with the prototypical u1 tracer ( +)- [3H]SKF-10,047.

MATERIALS AND METHODS Animals

For in vitro binding studies, male Sprague-Dawley rats (Iffa-Credo, L' Arbresles, France), weighing 200- 250 g, were used. For in vivo binding studies, Swiss mice were provided by the breeding center of the Faculty of Pharmacy (Montpellier, France). Male mice were used for competition experiments at 4-5 weeks of age and weighed 22-28 g. Non-pregnant and 14-day preg- nancy female mice were also used at 6-7 weeks of age.

736 Maurice et al.

TABLE I. Potencies of Neurosteroids in Inhibiting the In Vitro ( + )-[3H]-SKF-10,047 Binding in Rat Brain Homogenates*

Ki (nM) Compound Rat brain Guinea pig braina

PROG 175 * 55 268 +- 20 PREGS N.D. 3,196 ? 823 DHEA 2,959 t 134 N.D. DHEAS 15,126 t 7,696 N.D.

*Values are means f SEM of at least 3 determinations. N.D., not determined. aFrorn Su et al. (1988).

1993; Maurice et al., 1996b). Mice were injected intra- venously (IV) with 5 pCi ( +)-[3H]SKF-10,047 in 100 pl saline solution, and sacrificed 30 min later by cervical dislocation. The hippocampi and one cerebral cortex were dissected out, homogenized in 3 and 6 ml, respec- tively, of 5 mh4 Tris-HC1 pH 7.4 buffer at 4°C. Two 1 ml aliquots were filtered under vacuum through GF/B fil- ters, presoaked in 0.05% polyethyleneimine. The bound and total radioactivity were determined by counting two 200 ~1 aliquots of the homogenates. Non-specific bind- ing was defined as the remaining radioactivity after in- jection of haloperidol (2 mg/kg IP), 30 min before the tracer. Neurosteroids were administered SC, 10 min be- fore the tracer. Protein concentration was determined on 200 pl aliquots by the method of Bradford (1976).

Calculations and Statistical Analysis Results are expressed as mean & SEM. ICso values

were calculated by linear regression after log-probit transformation of data from competition experiments with at least 6 concentrations (Tallarida and Murray, 198 1). Ki values were calculated from the Cheng-Prussof equation (Cheng and Prussof, 1973). In vivo binding parameters were expressed as amount of ( +)-[3H]SKF- 10,047 bound, in fmol/mg of protein, and as bound to free radioactivity ratios (B/F ratios), as previously de- tailed (Maurice et al., 1996b). Data were analyzed using the Newman-Keuls’ multiple comparisons test after a two-way analysis of variance (ANOVA).

RESULTS In preliminary experiments, we confirmed that

PROG appears as a potent inhibitor of the in vitro (+)- [3H]SKF-10,047 binding to u1 sites on rat brain homoge- nates, with a Ki value of approximately 175 nM (Table I). None of the other neurosteroids tested presented a Ki value lower than 3 pM. Moreover, DHEAS appeared as the worst inhibitor, with a Ki value higher than 15 pM (Table I).

Parameters of in vivo ( +)-[3H]SKF-10,047 bind- ing to u, sites were determined in the hippocampal for- mation and cerebral cortex of the mice. Control animals showed total amounts of bound ( +)-[3H]SKF-10,047 similar in both structures, in the 10-13 fmol/mg of pro- tein range. These levels corresponded to B/F ratios in the 58-78% range. After injection of haloperidol (2 mg/kg IP), the levels of ( +)-[3H]SKF-10,047 binding de- creased to 0.6-1.4 fmol/mg, yielding a percentage of specific binding of about 93%. In parallel, the B/F ratios decreased to 4-7%.

As shown in Figure 1, the systemic administration of PROG, in the 2-40 mg/kg range, affected only weakly the amounts of bound ( +)-[3H]SKF-10,047. In the hippocampus, PROG displaced 39% of the specific binding at the highest dose, a decrease that appeared significant (P < 0.05; Fig. 1A). At the same dosage, however, a 32% decrease was observed in the cortex that remained nonsignificant compared to the control group (Fig. 1B). The B/F ratios led, however, to more marked differences: significant decreases were observed at 10, 20, and 40 mg/kg doses, corresponding to -36% in the hippocampus (Fig. IC) and -39% in the cortex (Fig. 1D).

The administration of PREGS, in the 10-40 mg/kg dose range, also induced dose-dependent decreases of in vivo ( +)-[3H]SKF-10,047 binding in both structures. At the highest dose, a significant 37% decrease in specific binding was observed in the hippocampus (P < 0.05; Fig. 2A) and a significant 44% decrease was observed in the cortex (P < 0.05; Fig. 2B). Regarding B/F ratios in the hippocampus, a significant 30% decrease was mea- sured at 40 mg/kg (P < 0.01; Fig. 2C). In the cortex, more marked differences were observed, since all the doses of PREGS significantly affected the B/F ratios, leading to a 26% decrease at 40 mg/kg (Fig. 2D).

DHEAS did not affect, in the 10-40 mg/kg dose range, the total ( + )-[3H]SKF-10,047 binding levels in either the hippocampus (Fig. 3A) or cortex (Fig. 3B). However, a significant 20% decrease in B/F was mea- sured in the hippocampus at the highest dose (Fig. 3C), but such an effect could not be observed in the cortex (Fig. 3D).

The in vivo ( +)-[3H]SKF-10,047 binding param- eters were also investigated in pregnant female mice, since important levels of PROG, as well as allopreg- nanolone levels are markedly increased during preg- nancy (Su et al., 1988; Paul and Purdy, 1992). Two- week pregnancy females were used, and the (+)- [3H]SKF- 10,047 binding was compared to non-pregnant females and males. As shown in Figure 4, the in vivo ( + )-[3H]SKF-10,047 binding parameters are signifi- cantly affected during pregnancy. The total amounts of ( + )-[3H]SKF-10,047 bound were significantly reduced

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Fig. 1. Dose-dependent effect of PROG on the in vivo ( + )- [3H]SKF-10,047 binding to u1 receptor sites in the mouse brain. Total (+)-['H]SKF-10,047 binding levels in the hippo- campus (A) and cortex (B), and BIF ratios in the hippocampus (C) and cortex (D). PROG was administered SC 10 min before

and In Vivo Binding to

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DOSES (mgkg)

by 32% in the hippocampus (Fig. 4A) and by 38% in the cortex (Fig. 4B) compared to non-pregnant females, whereas the B/F ratios were significantly decreased by 29% in the hippocampus (Fig. 4C) and by 31% in the cortex (Fig. 4D). In parallel, the amount of (+)- [3H]SKF- 10,047 bound and B/F ratios appeared strictly similar between non-pregnant female and male mice.

u1 Sites

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the tracer and haloperidol IP 30 min before the tracer. The numbers of animals are indicated within the columns in A. Veh, vehicle for PROG: sesame oil. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the Veh-treated group (Newman-Keuls' test).

DISCUSSION The present results show that several neurosteroids,

namely PROG, PREGS , and DHEAS , dose-dependently inhibited the in vivo binding of the prototypical u1 ra- diotracer ( +)-[3H]SKF-10,047 in the mouse hippocam- pus and cortex. Both parameters of the in vivo (+)- [3H]SKF-10,047 binding, i.e., the total amount of tracer

738 Maurice et al.

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Fig. 2 . Dose-dependent effect of PREGS on the in vivo ( +)- [3H]SKF-10,047 binding to uI sites in the mouse brain. Total ( + )-[3H]SKF-10,047 binding levels in the hippocampus (A) and cortex (B), and BIF ratios in the hippocampus (C) and cortex (D). PREGS was administered SC 10 min before the

bound expressed in fmoYmg of protein, and the B/F ra- dioactivity ratios, were affected although differentially among the doses and compounds. PROG appeared to be the most active neurosteroid, whereas DHEAS was the least potent, in complete accordance with their respective in vitro affinities.

As previously reported by others (Fems et al., 1988; Tam et al., 1988; Weissman et al., 1990; Bouchard

*

tracer and haloperidol IP 30 min before the tracer. The num- bers of animals are indicated within the columns in A. Veh, vehicle for PREGS: dimethylsulfoxide. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the Veh-treated group (Newman- Keuls’ test).

et al., 1993), ( +)-[3H]SKF-10,047 administered IV at a tracer dose (5-10 pCi per mouse) allows a selective labeling of the u1 sites in forebrain structures, such as the hippocampal formation and cortex. The u1 sites are most abundant in these regions, and although ( + )-SKF- 10,047 is known to label the NMDA/phencyclidine sites with a similar affinity as the u1 receptors in vitro, only the latter appears to be sensitive to haloperidol. It is

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Neurosteroids and In Vivo Binding to a, Sites

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Fig. 3. Dose-dependent effect of DHEAS on the in vivo ( +)- [3H]SKF-10,047 binding to u1 sites in the mouse brain. Total (+ )-[3H]SKF-10,047 binding levels in the hippocampus (A) and cortex (B), and B/F ratios in the hippocampus (C) and cortex (D). DHEAS was administered SC 10 min before the

unclear at present if the in vivo ( +)-[3H]SKF-10,047 binding corresponds to a single class of u1 sites. Indeed, ( + )-SKF-10,047, like ( + )-pentazocine, appeared to bind with a high affinity and selectivity to u1 compared to u2 sites (Quirion et al., 1992), but recent evidence suggested that ul sites may not correspond to a single population. In particular, Monnet and Baulieu (1995),

tracer and haloperidol IP 30 min before the tracer. The num- bers of animals are indicated within the columns in A. Veh, vehicle for DHEAS: dimethylsulfoxide. *P < 0.05, ***P < 0.001 vs. the Veh-treated group (Newman-Keuls' test).

using in vitro [3H]norepinephrine release measures, and Debonnel et al. (1995), using in vivo extracellular re- cording and microiontophoresis, reported in a conver- gent manner that the effects induced by the novel u1 selective agonists BD-737, S-21377, S-21272, and JO- 1784 appeared to be differentially sensitive to the com- pounds usually considered u antagonists, i.e., haloperi-

740 Maurice et al.

Hippocampus cortex

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Fig. 4. In vivo binding parameters of (+ j-[3H]SKF-10,047 to u1 sites in pregnant and non-pregnant females and males mice. Total ( + )-[3H]SKF-10,047 binding levels in the hippocampus (A) and cortex (B), and B/F ratios in the hippocampus (C) and

do1 , reduced haloperidol, NE- 100, BD- 1008, and S-20955. These authors speculated in favor of the poten- tial existence of subtypes of u1 sites. Thus, it must be kept in mind that although no radiotracer exists yet which allows a more selective in vivo labeling of the u1 recep- tors than ( +)-[3H]SKF-10,047 (Bouchard et al., 1993), the exact nature or subtype of u1 sites labeled by the drug remains to be delineated.

In this report, as well as in previous ones (Maurice and Vignon, 1990; Maurice et al., 1996b), we presented B/F ratios, in parallel with the total levels of bound tracer

D

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cortex (D). The numbers of animals are indicated within the columns in A. *P < 0.05 vs. the non-pregnant females group (Newman-Keuls' test).

expressed as fmol/mg, as usually reported by others (Fer- ris et al., 1988; Bouchard et al., 1993, 1995). The B/F ratios allow a more accurate measurement of the fraction of tracer bound to the site in vivo, compared to the total amount of tracer available in the brain structure, i.e., the bound plus free radioactivity amount. Artifactual prob- lems, ranging from mild differences in the quantity of tracer administered IV to metabolic effects induced by steroids on the blood-brain barrier permeability, could thus be avoided. Indeed, PROG, PREGS, or DHEAS did not significantly affect the total levels of free radioactiv-

Neurosteroids and In Vivo Binding to u1 Sites 741

onists haloperidol and NE-100, as well as by PROG. Finally, we reported that DHEAS, at doses of approxi- mately 10-20 mgkg, significantly attenuated the dizo- cilpine-induced impairment of learning, evaluated using spontaneous alternation in the Y-maze for spatial work- ing memory and step-down type of passive avoidance for long-term memory (Maurice et al., 1996a). The anti- amnesic effect of DHEAS, similar to those of other u1 agonists, was antagonized by BMY-14,802 and sup- pressed by a subchronic treatment with haloperidol dur- ing 7 days, clearly indicating an interaction with u, re- ceptors. These studies, together with the in vivo binding profiles reported in this study, extend the in vitro binding results by confirming that neurosteroids, and particularly PROG, are potent u1 system modulators, and by indi- cating that the in vivo interaction seems more pro- nounced than suggested by the in vitro affinities. Some discrepancies appeared, however, since PREG and PREGS did not modify the NMDA-induced activity of the pyramidal CA, neurons (Bergeron et al., 1996), al- though the latter inhibited the ( +)-[3H]SKF-10,047 la- beling.

It must be noted that none of the steroids tested in this study displaced more than 40% of the ( +)-[,H]SKF- 10,047 labeling, while haloperidol competed for more than 90% of the binding in both regions. Furthermore, the B/F ratio profiles observed for PROG or PREGS in the cortex suggested that the inhibition reached a plateau value. This may not be due to the selected dose range, since we and others reported that PROG, PREG, DHEA, and their sulfate esters exerted their behavioral effects at this precise dosage (Roberts et al., 1987; Flood and Rob- erts, 1988; Flood et al., 1992; Maurice et al., 1996a) through a a,-related mechanism (Maurice et al, 1996a; submitted material). Furthermore , similar profiles were observed by Bouchard et al. (1993, 1995) with neuro- peptide Y (NPY) or calcitonin gene-related peptide (CGRP), which displaced only one-third of the in vivo ( + )-[3H]SKF-10,047 labeling. These observations strongly suggest that the radiotracer could interact with different subtypes of u1 sites, neurosteroids, and/or NPY- and CGRP-related peptides interacting with only one subtype. However, the complex metabolism of ste- roids in the brain must also be considered, and more extensive studies will be performed to elucidate this point.

Interestingly, the in vivo binding parameters for ( +)-[3H]SKF-10,047 in pregnant female mice appeared markedly altered compared to non-pregnant ones. It was reported that PROG levels in human plasma ranged from 30 nM during the latter part of the menstrual cycle to approximately 450 nM in late pregnancy, which corre- sponded to an approximately 10-60% fractional occu- pancy at u, receptors labeled in vitro using (+)-

ity (data not shown), but results expressed as B/F led to more significant differences as demonstrated by examin- ing the levels of bound radioactivity, and they must be considered physiologically relevant.

In the first study, by Su et al. (1988), describing the inhibitory effect of steroids on the in vitro binding at u receptors, PROG appeared to be the most potent in- hibitor of ( +)-[3H]SKF-10,047 binding in guinea pig brain homogenates, with a Ki value of approximately 268 nM. We reported here a Ki value of about 175 nM in the rat brain. PREGS presented a Ki value of about 3.2 pM in the guinea pig brain (Su et al., 1988; Table I). Among the other steroids tested, testosterone and the adrenocor- tical steroid deoxycorticosterone appeared to be equipo- tent with a Ki value of approximately 1 pM (Su et al., 1988). We measured an affinity for DHEA of about 3 pM, but DHEAS appeared to be much more inactive. More recently, Klein and Musacchio (1994) examined the in vitro binding of various steroids on u sites labeled using [3H]dextrometorphan and ( + )-[,H]3-PPP. The most potent neurosteroid was PROG, with K, values of approximately 260 nM for ( +)-[,H]3-PPP binding and 650 nM for [3H]dextrometorphan binding (Klein and Musacchio, 1994). Among the other compounds tested, dihydrotestosterone, androsterone, testosterone, 5a-an- drostane-3 , 17-dione, and deoxycorticosterone presented K, values below 1 pM for (+>-[,H]3-PPP binding. PREG and DHEA appeared to be less efficient, with Ki values of approximately 7 and 3.7 pM, respectively, but their sulfate esters were not tested. In summary, the in vitro binding studies, using different u radioligands, led to convergent results, indicating that PROG displaced the ( +)-[3H]SKF-10,047, ( +)-[,H]3-PPP, or [,H]halo- peridol binding with Ki values in the low micromolar range (Su et al., 1988; Ross, 1991; Klein and Musac- chio, 1994). PREGS appeared to be less efficient, whereas DHEAS was inefficient.

Functional studies have subsequently demonstrated that PROG acts as a potent u1 antagonist. Furthermore, although PREGS or DHEAS presented only moderate affinities in vitro, effective interactions could be evi- denced in vivo and both neurosteroids acted as u, ago- nists. First, Monnet et al. (1995) reported that DHEAS potentiated whereas PREGS inhibited the NMDA- evoked release of [3H]norepinephrine from preloaded rat hippocampal slices, indicating that DHEAS acted as a u1 agonist and PREGS acted as a u1 inverse agonist in the test. Furthermore, PROG mimicked the antagonist effect of haloperidol by blocking the DHEAS, PREGS, and DTG agonist effects. On the other hand, Bergeron et al. (1996) reported that DHEA potentiated the NMDA- evoked electric activity of pyramidal neurons in the CA, region of the rat hippocampus, and thus behaved as a u, agonist, an effect that could be blocked by the m1 antag-

742 Maurice et al.

[3H]SKF-10,047 in brain homogenates (Su et al., 1988). A physiological confirmation came from the results by Bergeron et al. (1995, 1996), who reported that in preg- nant rats the u agonist DTG and DHEA were less effec- tive in potentiating the NMDA-evoked electric activity of pyramidal neurons in the CA, region of the rat hip- pocampus. A good correlation could thus be observed with the in vivo ( +)-[3H]SKF-10,047 binding, since 30-40% decreases could be measured in the hippocam- pus or cortex, confirming directly that modification of the physiological concentrations in PROG affect the u1 receptor occupancy.

Finally, the u1 receptors were recently purified and the cDNA cloned from guinea pig liver (Hanner et al., 1996). Its open reading frame encoded a 25.3 kDa pro- tein with at least one transmembrane segment, which revealed no homology to known mammalian proteins (Hanner et al., 1996). However, some homology was evidenced with fungal proteins involved in sterol synthe- sis, which gives some support to the interaction between neurosteroids and u1 receptor ligands.

In summary, these results established that neuro- steroids, such as PROG, PREGS, and DHEAS, do in- teract in vivo with u1 receptors after acute systemic ad- ministration. Furthermore, the increased PROG levels in the pregnant mouse led to a lowering of the (+)- [3H]SKF-10,047 binding parameters, confirming that the physiological modulations of the steroidal concentrations affect the u1 systems.

REFERENCES Bergeron R, De Montigny C, Debonnel G (1995): The potentiation of

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