Brain Research 799 1998.

6477

Research report

The modulation by neurosteroids of the scopolamine-induced learning impairment in mice involves an interaction with sigma 1 s 1 . receptors
Alexandre Urani, Alain Privat, Tangui Maurice
)

Unite 336 de lInstitut National de la Sante et de la Recherche Medicale, Deeloppement, Plasticite et Vieillissement du Systeme Nereux, ENSCM, 8, rue ` de lEcole Normale, 34296 Montpellier Cedex 5, France Accepted 21 April 1998

Abstract Neurosteroids have been reported to modulate learning and memory processes in aged animals and in pharmacological models of amnesia. We report here the effects of dehydroepiandrosterone sulfate DHEAS., pregnenolone sulfate PREGS., and progesterone PROG. on the learning impairment induced in mice by the muscarinic acetylcholine receptor antagonist, scopolamine. Spatial working memory was examined using the spontaneous alternation behavior in a Y-maze and long-term memory using place learning in a rectangular water-maze adapted for mice. Both DHEAS and PREGS 520 mgrkg, s.c.. prevented dose-dependently and significantly the scopolamine 2 mgrkg, s.c..-induced alternation deficits. PROG 220 mgrkg, s.c.. failed to affect the scopolamine-induced deficits, but blocked, at 20 mgrkg, the beneficial effects induced by DHEAS or PREGS. In the water-maze, DHEAS 20 mgrkg. attenuated significantly the scopolamine-induced deficits, as observed during the acquisition sessions or the retention test. PROG 2, 20 mgrkg. did not affect the control or scopolamine-treated group performances, but blocked the ameliorating effect of DHEAS. Furthermore, in both tests, the selective sigma 1 s 1 . receptor antagonist NE-100 1 mgrkg, i.p.. failed to affect the behaviors showed by the control or scopolamine-treated groups, but it blocked the ameliorating effects induced by DHEAS or PREGS. These results confirm the modulating role of neurosteroids in learning and memory processes and demonstrate that their modulation of the cholinergic systems involves an interaction with s 1 receptors. q 1998 Elsevier Science B.V. All rights reserved.
Keywords: Sigma 1 s 1 . receptor; Neuroactive neurosteroid; Scopolamine-induced amnesia; Y-maze; Water-maze; Learning and memory; Mouse

1. Introduction Neuroactive steroids play important roles in the central nervous system w55,59,68x. In particular, they exert a potent modulation of the learning and memory processes, as reported using several pharmacological and age-related models of amnesia w911,55x. The mechanism for this effect remains unclear, but it may be based on the facilitation or inhibition of several neurotransmitter systems, which are involved in the memory processes. Dehydroepiandrosterone sulfate DHEAS. and pregnenolone sulfate PREGS. were reported to interfere with the barbiturate-mediated enhancement of benzodiazepine binding, acting as a negative allosteric modulator of the g-aminobutyric acid A GABA A . receptor w2325x. Both neuros-

) Corresponding author. Fax: q33r0-4-6754-0610; E-mail: maurice@cit.enscm.fr

teroids also enhanced several N-methyl-D-aspartate NM DA . receptor-mediated responses in vitro w4,8,14,15,48,68x and in vivo w12,22x. In addition, DHEAS was recently shown to induce a dose-dependent release of acetylcholine in the rat hippocampus in vivo w58x. Consequently, DHEAS and PREGS have demonstrated memory-enhancing effects in amnesia models involving either blockade of the cholinergic neurotransmission w11,21,44x or of the NMDA-type of glutamatergic neurotransmission w5,26,27,38x. The exact mechanism of these neuromodulatory effects induced by neurosteroids is still controversial, but it appeared to be distinct from their genomic effects. PROG, PREGS, testosterone, and 17b-estradiol, among others, have been shown to significantly inhibit the in vitro binding of radioligands, that selectively label the sigma 1 s 1 . receptors, such as q.-w3 HxSKF-10,047, w3 Hxdextromethorphan, q.-w3 Hx3-PPP, or w3 Hxhaloperidol, from rat brain, splenocytes plasma membranes and liver micro-

0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.

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somes w19,45,62,66,70x. We recently reported that PROG, PREGS, and, in a lesser extent, DHEAS also affected the in vivo binding of q.-w3 HxSKF y 10,047 to s 1 sites in the mouse forebrain structures w40x. Furthermore, the levels of in vivo q.-w3 HxSKF-10,047 binding were significantly reduced in pregnant mice as compared to non-pregnant female or male, indicating that physiologic modulations of the steroidal levels affect s 1 receptors w40x. Such interaction with the s 1 receptor may constitute a possible mechanism for the steroidal non-genomic effects. Although initially postulated, s sites differ from opioid as well as NMDA receptor-associated phencyclidine binding sites w57x. The unique ligand specificity and autoradiographic distribution of s sites in both the central nervous system and peripheral tissues suggested that they belong to a distinct receptor family. Binding strategies, using in vitro or in vivo bioassays, provided evidence for the existence at least of two subtypes of s sites, denoted s 1 and s 2 w56x. The s 1 site was recently purified and the cDNA was cloned, from guinea-pig liver w13x and then from human placental choriocarcinoma cell w18x, with a 93% identity. The amino-acid sequences were structurally unrelated to known mammalian proteins, but the guinea-pig sequence shared a 66% homology with fungal sterol C 8 C 7 isomerase, which seemed in agreement with the putative link between steroids and the s 1 receptor w13x. Furthermore, s 1 receptor agonists induce a potent modulation of the cholinergic systems. In particular, they potentiated the acetylcholine release from the rat cortex and hippocampus in vitro and in vivo w16,20,2830x. They also showed anti-amnesic properties against the amnesia induced in rodents by muscarinic cholinergic receptor antagonists w6,3134x. Moreover, s 1 receptor ligands potentiated several NMDA-evoked responses, such as the NMDA-induced electric activity of rat dorsal hippocampal CA 3 pyramidal neurons or the NMDA-evoked w3 Hxnorepinephrine release from rat hippocampal slices w46,47,61x. They also attenuated the learning impairment induced by dizocilpine, a non-competitive NMDA receptor antagonist w7,34,36,37,42,50x. A similar pharmacology was observed between the effects of s 1 ligands and neurosteroids on the NMDA-mediated responses, DHEAS andror PREGS modulating the NMDA-evoked responses in a manner sensitive to s 1 receptor antagonists, while PROG behaved as an antagonist and blocked the effects of the s 1 nonsteroidal agonists w3,35,38,41,48x. No such similar pharmacology has been yet reported on the modulation of the cholinergic systems induced by neurosteroids. In this study, we examined the effects of DHEAS, PREGS and PROG on the learning impairments induced in mice by the muscarinic cholinergic receptor antagonist scopolamine, using the spontaneous alternation test and place learning in a water-maze. In addition, we investigated the effect of the selective s 1 receptor antagonist NE-100 w5153x on the ameliorating effects induced by the steroids.

2. Material and methods 2.1. Animals Male Swiss mice breeding center of the Faculty of Pharmacy, Montpellier, France., aged 56 weeks and weighing 3035 g were used. Animals were housed in plastic cages, with free access to laboratory chow and water, except during behavioral experiments, and kept in a regulated environment 23 " 18C, 4060% humidity., under a 12-h lightrdark cycle light on at 8:00 h.. Experiments were carried out between 10:00 h and 6:00 h, in a soundproof and air-regulated experimental room, to which mice were habituated at least 30 min before each experiment. Animal care followed the protocols and guidelines approved by INSERM. 2.2. Drugs and administration procedures N , N -dipropyl-2- w 4-m ethoxy-3- 2-phenylethoxy .phenylxethylamine hydrochloride NE-100. was supplied by Taisho Pharmaceutical Tokyo, Japan.; scopolamine hydrobromide, dehydroepiandrosterone sulfate 5-androsten-3 b-ol-17-one sulfate, DHEAS., pregnenolone sulfate 5-pregnen-3 b-ol-20-one sulfate, PREGS., and progesterone 4-pregnene-3,20-dione, PROG. were from Sigma St-Louis, MO, USA.. NE-100 was dissolved in distilled water. DHEAS was solubilized in dimethylsulfoxide DMSO, Sigma. and then in saline solution, final vehicle being DMSO 5% in saline. PREGS was solubilized in DMSO and then in distilled water, final vehicle being DMSO 5% in water. PROG was suspended in sesame oil Sigma.. Other compounds were dissolved in saline. Drugs were injected subcutaneously s.c.. or intraperitoneally i.p.., in a volume of 100 m lr20 g b.wt. Dose-ranges and administrations routes were selected according to the previous related studies w79,18,28,32,33,35,37,38x. 2.3. Spontaneous alternation performances Spatial working memory performance was assessed by recording spontaneous alternation behavior in a Y-maze w1,2,36,37,39,54,63x. The maze was made of black painted wood. Each arm was 40 cm long, 13 cm high, 3 cm wide at the bottom, 10 cm wide at the top, and converged at an equal angle. Each mouse was placed at the end of one arm and allowed to move freely through the maze during an 8 min session. The series of arm entries, including possible returns into the same arm, was recorded using an Apple IIe computer. An alternation was defined as entries into all three arms on consecutive occasions. The number of maximum alternations was therefore the total number of arm entries minus two and the percentage of alternation was calculated as actual alternationsrmaximum alternations. = 100. The drugs were administered 30 min before the session.

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2.4. Place learning in the water-maze test Place learning was examined using the water-maze test, as previously described w39,69x. The apparatus consisted in a black plexiglass rectangular pool 30 = 60 = 36 cm high., filled with water at a high of 25 cm. Skimmed milk powder was used to render the water opaque. The water temperature was maintained at 26 " 28C, with a bath heater, used before sessions. A transparent plexiglass platform 5 = 8.5 = 0.5 cm. was fixed in the southeast corner of the pool, 1 cm below the water surface. On days 1 and 2, training trials in the water-maze started 30 min after drug administrations. Each mouse was placed in the middle of the west side of the pool, facing the wall, and the latency spent to find the platform was recorded up to 60 s. The mouse was allowed to remain on it for 30 s, and then gently removed from the pool to its home cage. If the animal failed to find the platform within 60 s, the latency was assigned to 60 s, and it was manually placed on it for 30 s. Each animal was run in this manner for a total of 5 trials on day 1 and 3 trials on day 2. Inter-trials time interval was about 15 min. The starting and platform locations did not change during all training sessions. On day 4, 48 h after the last training session and the last drug injection, the animals were tested for retention. The platform was removed. Each mouse was placed again at the starting position and observed for 60 s. The latency to reach the initial position of the platform and the time spent within this area were recorded.

2.5. Statistical analysis Results are expressed as means " S.E.M. Alternation percentages were analyzed using the Dunnetts multiple comparison test after a one-way analysis of variance. Latencies failed to show a normal distribution, since cut-off times were set. They were analyzed over trials using the Friedman repeated measures nonparametric test, group comparisons being analyzed using the KruskalWallis nonparametric analysis of variance, followed by the Dunns nonparametric multiple comparisons test. The level for statistical significance was P - 0.05.

3. Results 3.1. Effects of neurosteroids on the scopolamine-induced spontaneous alternation deficits in mice The effects of DHEAS, PREGS and PROG were first examined on the spontaneous alternation behavior in the Y-maze. Each neurosteroid administered alone, in the 520 mgrkg s.c. dose-range failed to affect the exploratory behavior of untreated mice in the Y-maze, as previously reported w35,38,41x. Administration of scopolamine 2 mgrkg, s.c.. induced a marked decrease in spontaneous alternation black columns, Fig. 1. compared with control animals white columns., with no effect on the number of arms entered during the 8-min session. The simultaneous

Fig. 1. Effects of neurosteroids on spontaneous alternation deficits induced by scopolamine in mice: A. DHEAS, B. PREGS, and C. PROG. DHEAS 520 mgrkg., PREGS 520 mgrkg., and PROG 220 mgrkg. were administered 10 min before scopolamine 2 mgrkg., which was given 20 min before the test. Each value shows mean " S.E.M. of the number of animals indicated below each column. Total number of arm entries did not differ significantly across groups and were in the 2430 range in A., in the 2430 range in B., and in the 2429 range in C.. Sal: saline solution; Veh: vehicles were DMSO 5% in saline for DHEAS, DMSO 5% in water for PREGS, and sesame oil for PROG. ) P - 0.05, )) P - 0.01 vs. the control group; aP - 0.05 vs. the scopolamine-treated group Dunnetts test..

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Fig. 2. Antagonism by PROG of the neurosteroidal effects on the spontaneous alternation deficits induced by scopolamine in mice: A. DHEAS, B. PREGS. PROG 2, 20 mgrkg. was administered simultaneously with DHEAS 20 mgrkg. or PREGS 20 mgrkg., 10 min before scopolamine 2 mgrkg., which was given 20 min before the test. Each value shows mean " S.E.M. of the number of animals indicated below each column. Total number of arm entries did not differ significantly across groups and were in the 2632 range in A., and in the 2732 range in B.. Sal: saline solution; Veh: vehicles were DMSO 5% in saline for DHEAS, DMSO 5% in water for PREGS, and sesame oil for PROG. )) P - 0.01 vs. the control group; aP - 0.05 vs. the scopolamine-treated group; o P - 0.05 vs. the DHEASq scopolamine.- or PREGSq scopolamine.-treated group Dunnetts test..

administration of DHEAS or PREGS, led to a dose-dependent attenuation of the scopolamine-induced deficit in alternation Fig. 1A,B., significant differences being observed with the Veh q scopolamine.-treated group at the 20 mgrkg dosage. PROG, however, failed to affect the scopolamine-induced decrease in alternation Fig. 1C.. When PROG 2, 20 mgrkg. was administered simultaneously with either DHEAS Fig. 2A. or PREGS Fig. 2B., each at 20 mgrkg, a significant blockade of their respective attenuating effects could be observed at the highest dose of PROG Fig. 2A,B.. PROG behaved thus as an antagonist of the attenuating effects of DHEAS or PREGS on the scopolamine-induced spontaneous alternation deficits. 3.2. Effects of DHEAS on the scopolamine-induced deficits in place learning in the water-maze Place learning in the water-maze was analyzed during acquisition session in term of escape latencies. The mean latencies spent to reach the platform decreased over the training sessions for the control Veh q Sal.-treated group P - 0.01, Friedmans test, Fig. 3A.. On the contrary, the latencies exhibited by the Veh q scopolamine.-treated group did not decrease significantly P ) 0.05, Fig. 3A.. Furthermore, all latencies appeared significantly higher than those compared with the control group. The results indicated that control animals tended to learn correctly the

platform location, whereas scopolamine-treated animals did not. Animals treated with DHEAS, 20 mgrkg s.c. 30 min before the first training session on each day, showed an acquisition profile similar to control animals, the latencies decreasing significantly P - 0.01, Fig. 3A., with no significant difference as compared with controls. For the DHEASq scopolamine.-treated group, the latencies decreased P - 0.01. over training sessions. Moreover, the latencies measured during the two last swims appeared significantly different from those showed by the Veh q scopolamine.-treated group. The effects of the different treatments on learning could be evidenced during the retention session, as shown in the lower panel of Fig. 3. Two days after the last training session, the platform was removed and the animals were allowed a 60-s swim. The control animals showed a latency to reach the initial platform location of 9 " 2 s Fig. 3B., and they remained within this area about 15 " 2 s Fig. 3C.. The group treated with scopolamine during training exhibited a significantly increased latency of 24 " 4 s P - 0.01, Fig. 3B.. In parallel, the time spent within the platform location was decreased to 4 " 1 s P - 0.01, Fig. 3C.. The DHEAS treatment did not affect the retention parameters as compared with control animals. For the group treated with DHEASq scopolamine. during training, a non-significant attenuation in latency Fig. 3B., together with a significant increase in time Fig. 3C. were observed as compared to the Veh q scopolamine.-treated

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Fig. 3. Acquisition profiles A. and retention parameters B, C. in the water-maze test for mice treated with DHEAS and scopolamine. DHEAS 20 mgrkg, s.c.. and scopolamine 2 mgrkg, s.c.. were administered 30 min before the first acquisition trial on each training day. Retention was measured 48 h after the last training with the platform removed. The mean latency to reach the initial platform location B. and the total time spent within this area C. were recorded. Veh: vehicle, DMSO 5% in saline; Sal: saline; Scop: scopolamine. The numbers of animals per group were: n s 19 for the Veh q Sal.and Veh q Scop.-treated groups, n s 18 for the DHEASq Sal.-treated group, and n s 32 for the DHEASq Scop.-treated group. ) P - 0.05, )) P - 0.01 vs. the Veh q Sal.-treated group; aP - 0.05, aaP - 0.01 vs. the Veh q Scop.-treated group Dunns test..

group. Only the latency remained significantly different from the parameters showed by controls. These results indicated clearly that DHEAS allows a significant improvement of the learning deficits induced by scopolamine in the water-maze test. The acquisition profiles obtained after treatment with PROG, 2 or 20 mgrkg, are summarized in Fig. 4A,B. The control Veh q Sal.-treated group showed a significant decrease in latencies over training sessions P - 0.05, Fig. 4A.. For the Veh q scopolamine.-treated group, no significant decrease was observed P ) 0.05, Fig. 4A.. Moreover, the latencies measured in each trial, with an excep-

tion for trial 7, appeared significantly higher than those showed by control animals. The PROG treatment, at 2 mgrkg Fig. 4A., did not affect the profiles showed by control or scopolamine-treated animals. The PROGq Sal.-treated group exhibited a significant decrease in latencies P - 0.01., with no difference as compared with the control group. The PROGq scopolamine.-treated group showed no significant decrease in latencies P ) 0.05., with significant differences as compared with controls, particularly during the second acquisition day. The PROG treatment, at 20 mgrkg, affected moderately the profiles showed by control or scopolamine-treated animals Fig.

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Fig. 4. Acquisition profiles A, B. and retention parameters C, D. in the water-maze test for mice treated with PROG, 2 mgrkg A. or 20 mgrkg B. and scopolamine. PROG 2, 20 mgrkg, s.c.. and scopolamine 2 mgrkg, s.c.. were administered 30 min before the first acquisition trial on each training day. Retention was measured 48 h after the last training with the platform removed. The mean latency to reach the initial platform location C. and the total time spent within this area D. were recorded. Veh: vehicle, sesame oil; Sal: saline; Scop: scopolamine. The numbers of animals per group were: n s 14 for the Veh q Sal.-and Veh q Scop.-treated groups, n s 15 for the PROG 2 q Sal.-, PROG 2 q Scop.-, PROG 20 q Sal.- and PROG 20 q Scop.treated groups. ) P - 0.05, )) P - 0.01 vs. the Veh q Sal.-treated group; aP - 0.05 vs. the Veh q Scop.-treated group Dunns test..

4B.. The PROGq Sal.-treated group showed a significant decrease in latencies P - 0.05., with no difference with the control group. The PROG q scopolamine.-treated

group showed a significant decrease in latencies P 0.01., with significant differences as compared with the control group, particularly during the second acquisition

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Fig. 5. Acquisition profiles A, B. and retention parameters C, D. in the water-maze test for mice co-treated with PROG plus DHEAS, and scopolamine. PROG 2 mgrkg, s.c.., DHEAS 20 mgrkg, s.c.. and scopolamine 2 mgrkg, s.c.. were administered 30 min before the first acquisition trial on each training day. Retention was measured 48 h after the last training with the platform removed. The mean latency to reach the initial platform location C. and the total time spent within this area D. were recorded. The numbers of animals per group were: n s 18 for the Veh q Veh q Sal.-treated group, n s 20 and Veh q Veh q Scop.-treated group, n s 17 for the PROGq Veh q Sal.-treated group, n s 18 for the PROGq Veh q Scop.-treated group, n s 19 for the Veh q DHEASq Sal.-treated group and n s 20 for the PROGq DHEASq Scop.-treated group. ) P - 0.05, )) P - 0.01 vs. the Veh q Veh q Sal.-treated group; aP - 0.05, aaP - 0.01 vs. the Veh q Veh q Scop.-treated group; o P - 0.05, oo P - 0.01 vs. the Veh q DHEASq Scop.-treated group Dunns test..

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day. The latencies measured on trial 6, but not on trials 7 and 8, appeared significantly lower P - 0.05. as compared with the scopolamine-treated animals. During the retention test, it appeared clearly that the PROG treatments did not affect the scopolamine-induced learning deficits in the water-maze. None of the PROG treatments, 2 or 20 mgrkg, affected the retention parameters showed either by the control Veh q Sal.-treated animals or by the Veh q scopolamine.-treated group during the retention test, both in terms of latency to reach the initial platform location Fig. 4C. or time spent within this area Fig. 4D.. 3.3. Antagonism by PROG of the DHEAS effect The putative antagonist effect of PROG was checked against the attenuating effect of DHEAS, 20 mgrkg, on the scopolamine-induced impairment of place learning. Fig. 5A,B summarizes the acquisition profiles obtained using a 2 mgrkg PROG treatment. As previously observed, the control Veh q Veh q Sal.-treated group showed a correct learning, since the latencies decreased over training trials P - 0.01, Fig. 5A.. The Veh q Veh q scopolamine.-treated group also showed some decrease in latencies P - 0.05, Fig. 5A.. However, significant differences were observed as compared with controls, particularly for the latencies measured from trials 5 to 8. As previously reported, the PROG treatment, at 2 mgrkg,

failed to affect the acquisition profiles of controls or scopolamine-treated animals Fig. 5A.. For the PROGq Veh q Sal.-treated group, the latencies decreased P 0.01, Fig. 7A.. The PROGq Veh q scopolamine.-treated group showed no significant variation in latencies P ) 0.05, Fig. 5A., and the latencies measured from trials 5 to 8 appeared significantly higher than those measured for control animals. For the Veh q DHEASq scopolamine.treated group, the latencies decreased P - 0.01, Fig. 5B.. However, only the latency measured on trial 7 appeared significantly lower than the one measured for the scopolamine-treated animals. For the PROG q DHEAS q scopolamine.-treated group, no decrease in latencies was observed P ) 0.05, Fig. 5B., the latencies appearing significantly higher compared with the control group from trial 3 to 8, and on trial 4 and 7 compared with the Veh q DHEASq scopolamine.-treated group. The antagonist effect of PROG appeared clearly in the retention parameters, presented in the lower panel of Fig. 5. The PROG treatment did not affect each parameter as compared with control or scopolamine-treated animals. However, the non-significant attenuation of latency Fig. 5C. and the significant increase in time Fig. 5D. induced by the DHEAS treatment were blocked by PROG, in a significant manner as compared with the Veh q DHEAS q scopolamine.-treated group P - 0.01 each.. A similar antagonism of the DHEAS effect on the scopolamine-induced learning impairment could be ob-

Fig. 6. Antagonism by NE-100 of the neurosteroidal effects on the spontaneous alternation deficits induced by scopolamine in mice: A. DHEAS, B. PREGS. NE-100 1 mgrkg, i.p.. was administered simultaneously with DHEAS 20 mgrkg, s.c.. or PREGS 20 mgrkg, s.c.., 10 min before scopolamine 2 mgrkg, s.c.., which was given 20 min before the test. Each value shows mean" S.E.M. of the number of animals indicated below each column. Total number of arm entries did not differ significantly across groups and were in the 2631 range P ) 0.05. in A., and in the 2835 range P ) 0.05. in B.. Sal: saline solution; Veh: vehicles were water for NE-100, DMSO 5% in saline for DHEAS, and DMSO 5% in water for PREGS. )) P - 0.01 vs. the Veh q Veh q Sal.-treated group; aP - 0.05, aaP - 0.01 vs. the Veh q Veh q Scop.-treated group; 8P - 0.05 vs. the Veh q DHEASq Scop.-treated group Dunnetts test..

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Fig. 7. Acquisition profiles A, B. and retention parameters C, D. in the water-maze test for mice co-treated with NE-100 plus DHEAS, and scopolamine. NE-100 1 mgrkg, i.p.., DHEAS 20 mgrkg, s.c.. and scopolamine 2 mgrkg, s.c.. were administered 30 min before the first acquisition trial on each training day. Retention was measured 48 h after the last training with the platform removed. The mean latency to reach the initial platform location C. and the total time spent within this area D. were recorded. The numbers of animals per group were: n s 19 for the Veh q Veh q Sal.-treated group, n s 20 and Veh q Veh q Scop.-treated group, n s 14 for the NE-100q Veh q Sal.-treated group, and n s 15 for the NE-100q Veh q Scop.-, Veh q DHEAS q Sal.- and NE-100q DHEASq Scop.-treated groups. ) P - 0.05, )) P - 0.01 vs. the Veh q Veh q Sal.-treated group; aP - 0.05, aaP - 0.01 vs. the Veh q Veh q Scop.-treated group; o P - 0.05, oo P - 0.01 vs. the Veh q DHEASq Scop.-treated group Dunns test..

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served after a treatment with PROG at 20 mgrkg data not shown.. 3.4. Antagonism by NE-100 of the neurosteroidal effects The effect of the simultaneous administration of the selective s 1 receptor antagonist NE-100 was investigated on the attenuating effects of neurosteroids using each behavioral test. As shown in Fig. 6A, NE-100, at 1 mgrkg i.p., failed to affect the scopolamine-induced alternation deficits in the Y-maze. The drug, however, blocked the attenuating effects of DHEAS Fig. 6A. and PREGS Fig. 6B., the neurosteroids being administered at 20 mgrkg and all resulting differences appearing significant. In the water-maze test, the putative antagonist effect of NE-100 was examined on the improvement by DHEAS of the scopolamine-induced deficits. Fig. 7A,B summarizes the acquisition profiles observed after treatment with NE100 at 1 mgrkg andror DHEAS at 20 mgrkg. As already observed, the control Veh q Veh q Sal.-treated group showed a correct learning, with latencies decreasing significantly P - 0.01, Fig. 7A.. The Veh q Veh q scopolamine.-treated group also showed some decrease in latencies P - 0.05, Fig. 7A.. However, all latencies appeared significantly higher than those measured for control animals. The NE-100 treatment did not affect the profiles of control or scopolamine-treated animals Fig. 7A.. For the NE-100q Veh q Sal.-treated group, the latencies decreased P - 0.01. similarly as for controls. The NE-100 q Veh q scopolamine.-treated group showed no significant variation in latencies P ) 0.05., and all latencies appeared significantly higher than those measured for control animals. For the Veh q DHEAS q scopolamine.treated group, the latencies decreased over sessions P 0.01, Fig. 7B., with significant differences as compared with the scopolamine-treated group, from trial 3 to 8 excepting trial 7 Fig. 7B.. For the NE-100q DHEASq scopolamine.-treated group, the latencies also decreased over training trials P - 0.01, Fig. 7B.. However, latencies appeared significantly higher than those measured for the Veh q DHEASq scopolamine.-treated group from trials 2 to 5 and during trial 7. The NE-100 treatment failed to affect each retention parameter as compared with controls or scopolaminetreated animals Fig. 7C,D.. However, the significant attenuation of latency to reach the platform location Fig. 7C. and increase in time spent with this area Fig. 7D., induced by the DHEAS treatment, were blocked by NE-100 in a significant manner P - 0.01 each..

4. Discussion We investigated, in this study, the modulation by neurosteroids of the scopolamine-induced learning deficits in

mice using both a short-term memory test, the spontaneous alternation behavior in the Y-maze w1,2,36,37,54,63x, and a long-term memory test, place learning in a water-maze w39,69x. In accordance with previous studies w36,42x, the alternation percentage in the Y-maze was decreased in scopolamine-treated animals from 70% to 50%, the chance level, without any change in the number of arm entries. In parallel, place learning in the water-maze was markedly affected. During acquisition sessions, the latencies spent to find the platform did not significantly decrease over training trials: the repeated measures ANOVA did not reach significance or the post-hoc comparison test did not show significant differences between trials. Furthermore, latencies appeared significantly higher than the ones measured for the control group. Then, during the retention session performed 48 h after the last training session, the parameters showed by the scopolamine-treated group were affected: a higher latency to reach the location of the platform during training and a lower time spent within that area were measured. Both tests thus allowed a quantitative measure of the scopolamine-induced learning deficits. None of the neurosteroids tested, in the 520 mgrkg dose range, affected by itself the spontaneous alternation behavior w35,38,41x or place learning in the water-maze this study.. Administration of DHEAS or PREGS in combination with scopolamine resulted in a dose-dependent attenuation of the scopolamine-induced deficits in both tests. In the Y-maze, the effects induced by the two steroids appeared of similar extent, with a significant attenuation observed at 20 mgrkg. PROG did not affect the scopolamine-induced alternation deficits, but behaved as an antagonist, by blocking the beneficial effects exerted by both neurosteroids in the Y-maze or by DHEAS in the water-maze. These results clearly confirmed the antiamnesic effects of neurosteroids in an amnesia model involving blockade of the cholinergic muscarinic neurotransmission. The cognitive enhancing effects of neurosteroids in rodents have been previously reported using several behavioral tests. Post-training central administration of DHEA or DHEAS improved the T-maze footshock active avoidance or step-down type passive avoidance behaviors in mice, compared to the vehicle-treated animals w11,60x. In an extensive study, the same authors confirmed that i.c.v. administration of PREG, PREGS, DHEA, DHEAS, and testosterone, but not PROG, improved active avoidance retention in mice w10x. More recently, Frye and Sturgis w12x described the memory enhancing effects of PREG, PREGS and most efficiently DHEAS and 5 a-pregnan-3 a-ol-20-one using female rats submitted to place learning in the Morris water-maze test or to a delayed non-matching-to-sample test in a Y-maze. We did not observe any memory enhancing effects of neurosteroids by themselves in our tests, but the effects previously reported could be observed in undertrained animals in the active avoidance test w10,11,60x or by using the Morris water-maze test, that allows the observation of learning improvement

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w12x. In the present study, experiments were designed in order to measure significant deficits after the scopolamine treatment together with a pronounced and rapid learning for control animals, on both tests. Such conditions impeded the observation of any memory enhancing effects of the neurosteroids alone. Interestingly, we observed that PROG antagonized the effects of either DHEAS or PREGS under a simultaneous administration protocol. It is likely that these molecules do not cross the bloodbrain barrier at the same rate. Furthermore, sulfated steroids injected peripherally presumably do not penetrate the brain, but may be converted to nonsulfated PREG of DHEA by microsomal sulfatase w17x. The free steroids readily penetrate the brain, where they can be metabolized again in sulphated steroids, which represent the most active form, by the effects of sulfotransferases. It is thus expected that the kinetics of PROG, which is not subjected to sulphatation, would be faster than the ones of PREGS or DHEAS, allowing a simultaneous administration protocol. Neurosteroids have also been reported to alleviate the learning impairments, due to aging or observed after pharmacological manipulations. DHEAS administered after training at 20 mgrkg s.c. improved T-maze footshock active avoidance in aged mice w9x. The DHEAS-treated 18and 24-month old mice showed an improved learning ability, similar as 2-month old mice. Neurosteroids also alleviated amnesia induced in rodents by blockade of either the cholinergic or glutamatergic neurotransmission. Flood et al. w11x observed that DHEAS completely counteracted the amnesia induced by anisomycin, an inhibitor of protein synthesis, or scopolamine in mice submitted to the footshock active avoidance test. Li et al. w21x reported that peripheral administration of DHEAS alleviated the scopolamine-induced step-through type passive avoidance deficits in mice. Furthermore, the steroid sulfatase inhibitor estrone-3-O-sulfamate administered alone enhanced retention in the passive avoidance test and its administration in combination with DHEAS potentiated the neurosteroidal effect w21x. Mayo et al. w44x reported that direct infusions into the nucleus basalis magnocellularis of PREGS or tetrahydroprogesterone enhanced or disrupted, respectively, the performances of rats submitted to a twotrial alternation task in a Y-maze. Finally, we observed in this study that peripheral administration of DHEAS or PREGS, but not PROG, counteracted the scopolamine-induced deficits of either spontaneous alternation behavior or place learning in a water-maze in mice. PROG behaved as an antagonist, by blocking the DHEAS or PREGS effects. These results demonstrated that neurosteroids, and particularly DHEAS and PREGS, potentiate the acetylcholine-dependent memory processes. This effect was observed after central or peripheral administration, and it could be amplified by the sulfatase inhibitor, demonstrating that the sulfated form may be the most active one w12x. Recently, Rhodes et al. w58x provided a biochemical evidence for a

direct effect of neurosteroids on the cholinergic systems. DHEAS induced a dose-dependent increase in hippocampal acetylcholine release, measured in vivo using intracerebral microdialysis in the anesthetized rat. Interestingly, in the previously reported studies, DHEAS administered peripherally showed its anti-amnesic effect at a similar optimal dosage of 20 mgrkg, a dose that increased significantly the hippocampal acetylcholine release in the study by Rhodes et al. w58x. Interestingly, neurosteroids have also been reported to alleviate the memory impairments induced by blockade of the NMDA-type of glutamatergic neurotransmission. PREGS blocked the passive avoidance deficits induced by the competitive NMDA receptor antagonists 3-".-2carboxypiperazin-4-yl.-propyl-1-phosphonic acid CPP. and D-2-amino-5-phosphonovalerate D-AP5. w26,27x. Then, Cheney et al. w5x reported that PREGS, infused i.v. in adrenalectomizedrcastrated rats, attenuated the amnesia induced by the non-competitive NMDA receptor antagonist dizocilpine, likely through an effect mediated by allopregnanolone w5x. Finally, we reported that DHEAS, administered systemically or centrally, attenuated the dizocilpine-induced deficits in spontaneous alternation and step-down type passive avoidance behaviors in mice w35,38x. Different mechanisms can be proposed in order to explain the anti-amnesic effects of DHEAS or PREGS. Flood et al. w10x proposed that the neurosteroid-induced improvement of memory may imply their genomic effects, through the modulation of the rates and amounts of transcription of immediate-early genes. The immediate-early genes induce a facilitation of translational processes leading to the synthesis of enzymes and proteins, some of them being involved during the consolidation phase of the memory process. On the other hand, both DHEAS and PREGS are known to modulate negatively the GABA A receptor complex, potentiating the binding of GABAergic agonists and benzodiazepines w2325x. It is well-known that benzodiazepines are amnesic in rodents through their modulation of the GABA A receptor, with GABAergic antagonists blocking and GABAergic agonists potentiating the memory impairing effects of benzodiazepines w49,67x. Furthermore, Mayo et al. w43,44x reported that neurosteroids infused into the nucleus basalis magnocellularis improved the cognitive ability of rats in a two-trial recognition task in the same manner as b-carbolines, which decrease the GABAergic neurotransmission. However, PREGS appeared active only after a post-training administration protocol, but not after a pre-training administration as observed for b-carbolines at low doses w43,44x. Neurosteroids thus induce, through their negative modulation of GABA A receptors, a disinhibition of the cholinergic neurons of the nucleus basalis magnocellularis projecting into the hippocampus, and concomittently an increase in excitatory inputs through their positive modulation of NMDA receptors, mediating the memory enhancing effects w58x.

A. Urani et al.r Brain Research 799 (1998) 6477

75

A third mechanism can be proposed from the present study, through the neurosteroidsrs1 receptor interaction. It was previously reported that neurosteroids potentiate several NMDA-mediated responses in vitro or in vivo, in the same manner as previously described for the s 1 receptor agonists, q .-N-allyl-normetazocine q .-SKF10,047 ., 1,3-di-2-tolyl.guanidine DTG . , q .-Ncyclopropylmethyl-N-methyl-1,4-diphenyl-1-1-ethyl-but-3en-1-ylamine hydrochloride JO-1784. w3,4648x. In particular, the steroidal effects were blocked by s 1 receptor antagonists, such as haloperidol, 1w2-3,4-dichlorophenyl.ethylx-4-methyl piperazine BD-1063., or NE-100. Conversely, PROG behaved as an antagonist, blocking the steroidal effects and the effects induced by the non-steroidal s 1 receptor agonists. Consequently, we observed that in the dizocilpine-induced amnesia model in mice the antiamnesic effect of DHEAS could be blocked by the s 1 receptor antagonist a-4-fluorophenyl.-4-5-fluoro-2pyrimidinyl.-1-piperazinebutanol BMY-14,802. w38x, and that PROG blocked the anti-amnesic effect of the selective s 1 receptor agonist 1-3,4-dimethoxyphenethyl.-4-3phenyl propyl.piperazine dihydrochloride SA4503. w35x. Moreover, s 1 receptor agonists regulate the cholinergic neurotransmission, as reported using in vitro or in vivo techniques. First, Siniscalchi et al. w65x reported that q.SKF-10,047 potentiated the electrically evoked acetylcholine release from guinea-pig brain slices. Second, Kobayhashi et al. w20x and Matsuno et al. w2830x demonstrated that several s 1 receptor agonists, q.-SKF-10,047, ".-pentazocine, DTG, q.-3-PPP, and SA4503 increased the extracellular acetylcholine levels in the rat frontal cortex and hippocampal formation, using in vivo microdialysis in the rat. The increases were blocked by haloperidol and correlated with the binding affinities to the q.w3 HxSKF-10,047-labeled s 1 sites, demonstrating the direct involvement of the s 1 receptors w29x. Furthermore, s 1 receptor agonists have been reported to antagonize the scopolamine- or p-chloroamphetamine-induced amnesia in rats and mice submitted to a passive avoidance test, after pre-training, post-training, or pre-retention administration w6,3133,64x. We observed here that the anti-amnesic effect induced by neurosteroids against the scopolamine-induced learning impairment could be completely blocked by the selective s 1 receptor antagonist NE-100, demonstrating a clear similar pharmacology between neurosteroids and s 1 system on the acetylcholine-dependent learning, as observed on the NMDA-dependent responses and memory processes. In summary, this study confirmed the anti-amnesic effects of neurosteroids, by demonstrating that DHEAS and PREGS prevented the scopolamine-induced amnesia in mice and suggested that part of their anti-amnesic effect involves an interaction with s 1 receptor. Neurosteroids may indeed induce their effects through different mechanisms of action, involving rapid genomic events, disinhibition through their antagonism on GABA A receptor com-

plexes, and potentiation of excitatory inputs through a facilitation of NMDA receptor activation and an agonist effect at s 1 receptor. The differential involvement of these mechanisms in the cognitive effects of steroids may depend on the physiologic steroidal levels, on the brain structure involved in the learning task, or on the administration procedure. In particular, the consequences and therapeutic opportunities of the interaction between neurosteroids and the s 1 receptor in age-related and neurodegenerative cognitive disabilities is currently under investigation.

Acknowledgements Thanks are due to Jean Bayle for elaborating the apparatus used for behavioral testing, and to Taisho Pharmaceutical Tokyo, Japan. for providing us NE-100. This work was supported by INSERM.

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