alexandre urani, alain privat and tangui maurice- the modulation by neurosteroids of the...

8/3/2019 Alexandre Urani, Alain Privat and Tangui Maurice- The modulation by neurosteroids of the scopolamine-induced lear

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.Brain Research 799 1998 6477

Research report

The modulation by neurosteroids of the scopolamine-induced learning .impairment in mice involves an interaction with sigma s receptors1 1

Alexandre Urani, Alain Privat, Tangui Maurice )

Unite 336 de lInstitut National de la Sante et de la Recherche Medicale, De eloppement, Plasticite et Vieillissement du Systeme Nereux, ENSCM, 8, `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 model . .amnesia. We report here the effects of dehydroepiandrosterone sulfate DHEAS , pregnenolone sulfate PREGS , and progeste

.PROG on the learning impairment induced in mice by the muscarinic acetylcholine receptor antagonist, scopolamine. Spatial wor

memory was examined using the spontaneous alternation behavior in a Y-maze and long-term memory using place learning .rectangular water-maze adapted for mice. Both DHEAS and PREGS 5 20 mgrkg, s.c. prevented dose-dependently and significantly

. .scopolamine 2 mgrkg, s.c. -induced alternation deficits. PROG 2 20 mgrkg, s.c. failed to affect the scopolamine-induced deficits .blocked, at 20 mgrkg, the beneficial effects induced by DHEAS or PREGS. In the water-maze, DHEAS 20 mgrkg attenu

.significantly the scopolamine-induced deficits, as observed during the acquisition sessions or the retention test. PROG 2, 20 mgrkg

not affect the control or scopolamine-treated group performances, but blocked the ameliorating effect of DHEAS. Furthermore, in b . .tests, the selective sigma s receptor antagonist NE-100 1 mgrkg, i.p. failed to affect the behaviors showed by the contro

1 1

scopolamine-treated groups, but it blocked the ameliorating effects induced by DHEAS or PREGS. These results confirm the modula

role of neurosteroids in learning and memory processes and demonstrate that their modulation of the cholinergic systems involve

interaction with s receptors. q 1998 Elsevier Science B.V. All rights reserved.1

.Keywords: Sigma s receptor; Neuroactive neurosteroid; Scopolamine-induced amnesia; Y-maze; Water-maze; Learning and memory; Mouse1 1

1. Introduction

Neuroactive steroids play important roles in the centralw xnervous system 55,59,68 . In particular, they exert a po-

tent modulation of the learning and memory processes, as

reported using several pharmacological and age-relatedw xmodels of amnesia 911,55 . The mechanism for this

effect remains unclear, but it may be based on the facilita-

tion or inhibition of several neurotransmitter systems,which are involved in the memory processes. Dehy-

.droepiandrosterone sulfate DHEAS and pregnenolone .sulfate PREGS were reported to interfere with the barbi-

turate-mediated enhancement of benzodiazepine binding,

acting as a negative allosteric modulator of the g-amino- . w xbutyric acid GABA receptor 2325 . Both neuros-A A

)

Corresponding author. Fax: q33r0-4-6754-0610; E-mail:

[email protected]

teroids also enhanced several N-methyl-D-aspar .N M D A r ec ep to r- m ed ia te d r es po ns es i n vw x w x4,8,14,15,48,68 and in vivo 12,22 . In addition, DHE

was recently shown to induce a dose-dependent releasw xacetylcholine in the rat hippocampus in vivo 58 . Con

quently, DHEAS and PREGS have demonstra

memory-enhancing effects in amnesia models involv

either blockade of the cholinergic neurotransmis

w x11,21,44 or of the NMDA-type of glutamatergic new xtransmission 5,26,27,38 . The exact mechanism of th

neuromodulatory effects induced by neurosteroids is

controversial, but it appeared to be distinct from t

genomic effects.

PROG, PREGS, testosterone, and 17b-estradiol, am

others, have been shown to significantly inhibit the in v

binding of radioligands, that selectively label the sigm . . w3 x w3 xs receptors, such as q - H SKF-10,047, H dex1

. w3 x w3 xmethorphan, q - H 3-PPP, or H haloperidol, from

brain, splenocytes plasma membranes and liver mi

0006-8 3 8 1 .00 q 1 8 Elsevier Science B.V. All ri hts reserved.


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

w xsomes 19,45,62,66,70 . We recently reported that PROG,

PREGS, and, in a lesser extent, DHEAS also affected the . w3 xin vivo binding of q - H SKF y 10,047 to s sites in1

w xthe mouse forebrain structures 40 . Furthermore, the levels . w3 xof in vivo q - H SKF-10,047 binding were significantly

reduced in pregnant mice as compared to non-pregnant

female or male, indicating that physiologic modulations ofw xthe steroidal levels affect s receptors 40 . Such interac-1

tion with the s receptor may constitute a possible mecha-1nism for the steroidal non-genomic effects. Although ini-

tially postulated, s sites differ from opioid as well as

NMDA receptor-associated phencyclidine binding sitesw x57 . 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 atw xleast of two subtypes of s sites, denoted s and s 56 .1 2

The s site was recently purified and the cDNA was1w xcloned, from guinea-pig liver 13 and then from humanw xplacental choriocarcinoma cell 18 , 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 C iso-8 7merase, which seemed in agreement with the putative link

w xbetween steroids and the s receptor 13 . Furthermore,1s receptor agonists induce a potent modulation of the1cholinergic systems. In particular, they potentiated the

acetylcholine release from the rat cortex and hippocampusw xin vitro and in vivo 16,20,2830 . They also showed

anti-amnesic properties against the amnesia induced in

rodents by muscarinic cholinergic receptor antagonistsw x6,3134 . Moreover, s receptor ligands potentiated sev-1eral NMDA-evoked responses, such as the NMDA-in-duced electric activity of rat dorsal hippocampal CA 3

w3 xpyramidal neurons or the NMDA-evoked H norepineph-w xrine release from rat hippocampal slices 46,47,61 . They

also attenuated the learning impairment induced by di-

zocilpine, a non-competitive NMDA receptor antagonistw x7,34,36,37,42,50 . A similar pharmacology was observed

between the effects of s ligands and neurosteroids on the1NMDA-mediated responses, DHEAS andror PREGS

modulating the NMDA-evoked responses in a manner

sensitive to s receptor antagonists, while PROG behaved1as an antagonist and blocked the effects of the s non-1

w xsteroidal agonists 3,35,38,41,48 . No such similar phar-macology 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 investi-

gated the effect of the selective s receptor antagonist1w xNE-100 5153 on the ameliorating effects induced by the

steroids.

2. Material and methods

2.1. Animals

Male Swiss mice breeding center of the Faculty.Pharmacy, Montpellier, France , aged 56 weeks

weighing 3035 g were used. Animals were housed

plastic cages, with free access to laboratory chow

water, except during behavioral experiments, and kept .regulated environment 23"18C, 4060% humidity ,

.der a 12-h lightrdark cycle light on at 8:00 h . Exp

ments were carried out between 10:00 h and 6:00 h, i

soundproof and air-regulated experimental room, to wh

mice were habituated at least 30 min before each exp

ment. Animal care followed the protocols and guideli

approved by INSERM.

2.2. Drugs and administration procedures

w N, N-dipropyl-2- 4-m e thoxy-3- 2-phe nyle th. x .yphenyl ethylamine hydrochloride NE-100 was supp .by Taisho Pharmaceutical Tokyo, Japan ; scopolam

hydrobromide, dehydroepiandrosterone sulfate 5-and.ten-3b-ol-17-one sulfate, DHEAS , pregnenolone sul

.5-pregnen-3b-ol-20-one sulfate, PREGS , and prog .terone 4-pregnene-3,20-dione, PROG were from Sig

.St-Louis, MO, USA . NE-100 was dissolved in disti

water. DHEAS was solubilized in dimethylsulfox .DMSO, Sigma and then in saline solution, final veh

being DMSO 5% in saline. PREGS was solubilized

DMSO and then in distilled water, final vehicle be

DMSO 5% in water. PROG was suspended in sesame .Sigma . Other compounds were dissolved in saline. Dr

.were injected subcutaneously s.c. or intraperitone .i.p. , in a volume of 100 mlr20 g b.wt. Dose-ranges

administrations routes were selected according to the pw xvious related studies 7 9,18,28,32,33,35,37,38 .

2.3. Spontaneous alternation performances

Spatial working memory performance was assessed

recording spontaneous alternation behavior in a Y-mw x1,2,36,37,39,54,63 . The maze was made of black pain

wood. Each arm was 40 cm long, 13 cm high, 3 cm w

at the bottom, 10 cm wide at the top, and converged at

equal angle. Each mouse was placed at the end of one and allowed to move freely through the maze during a

min session. The series of arm entries, including poss

returns into the same arm, was recorded using an Apple

computer. An alternation was defined as entries into

three arms on consecutive occasions. The number of m

mum alternations was therefore the total number of

entries minus two and the percentage of alternation w calculated as actual alternationsrmaximum alternatio

=100. The drugs were administered 30 min before

session.


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2.4. Place learning in the water-maze test

Place learning was examined using the water-maze test,w xas previously described 39,69 . 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 timeinterval 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 plat-

form 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. Alterna

percentages were analyzed using the Dunnetts mult

comparison test after a one-way analysis of varia

Latencies failed to show a normal distribution, since cut

times were set. They were analyzed over trials using

Friedman repeated measures nonparametric test, gr

comparisons being analyzed using the KruskalWanonparametric analysis of variance, followed by the Dun

nonparametric multiple comparisons test. The level

statistical significance was P-0.05.

3. Results

3.1. Effects of neurosteroids on the scopolamine-indu

spontaneous alternation deficits in mice

The effects of DHEAS, PREGS and PROG were f

examined on the spontaneous alternation behavior in Y-maze. Each neurosteroid administered alone, in the 5

mgrkg s.c. dose-range failed to affect the explora

behavior of untreated mice in the Y-maze, as previouw xreported 35,38,41 . Administration of scopolamine

.mgrkg, s.c. induced a marked decrease in spontane .alternation black columns, Fig. 1 compared with con

.animals white columns , with no effect on the numbe

arms entered during the 8-min session. The simultane

. . .Fig. 1. Effects of neurosteroids on spontaneous alternation deficits induced by scopolamine in mice: A DHEAS, B PREGS, and C PROG. DH . . . .520 mgrkg , PREGS 520 mgrkg , and PROG 220 mgrkg were administered 10 min before scopolamine 2 mgrkg , which was given 20

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 di . . .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; V

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 coa .group; P-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 . . . PREGS. PROG 2, 20 mgrkg was administered simultaneously with DHEAS 20 mgrkg or PREGS 20 mgrkg , 10 min before scopolamin

.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 num . .of arm entries did not differ significantly across groups and were in the 26 32 range in A , and in the 2732 range in B . Sal: saline solution;

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;a

P-o . . .vs. the scopolamine-treated group; P-0.05 vs. the DHEASq scopolamine - or PREGSq scopolamine -treated group Dunnetts test .

administration of DHEAS or PREGS, led to a dose-depen-

dent attenuation of the scopolamine-induced deficit in .alternation Fig. 1A,B , significant differences being ob-

.served with the Vehq 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 simultane-

. .ously with either DHEAS Fig. 2A or PREGS Fig. 2B ,

each at 20 mgrkg, a significant blockade of their respec-

tive 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 VehqSal -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 anim

did not. Animals treated with DHEAS, 20 mgrkg s.c

min before the first training session on each day, show

an acquisition profile similar to control animals, the lat .cies decreasing significantly P-0.01, Fig. 3A , with

significant difference as compared with controls. For .DHEASqscopolamine -treated group, the latencies

.creased P-0.01 over training sessions. Moreover,

latencies measured during the two last swims appeasignificantly different from those showed by the Ve

.scopolamine -treated group.

The effects of the different treatments on learning co

be evidenced during the retention session, as shown in

lower panel of Fig. 3. Two days after the last train

session, the platform was removed and the animals w

allowed a 60-s swim. The control animals showed atency to reach the initial platform location of 9"2 s

.3B , and they remained within this area about 15" .Fig. 3C . The group treated with scopolamine du

training exhibited a significantly increased latency of 24 .4 s P-0.01, Fig. 3B . In parallel, the time spent wit

the platform location was decreased to 4"1 s P-0.Fig. 3C . The DHEAS treatment did not affect the ret

tion parameters as compared with control animals. For .group treated with DHEASqscopolamine during tr

ing, a non-significant attenuation in latency Fig. 3 .together with a significant increase in time Fig. 3C w

.observed as compared to the Vehq scopolamine -trea


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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. .mgrkg, s.c. and scopolamine 2 mgrkg, s.c. were administered 30 min before the first acquisition trial on each training day. Retention was measure

. 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 were recorded. Veh: vehicle, DMSO 5% in saline; Sal: saline; Scop: scopolamine. The numbers of animals per group were: n s 19 for the Vehq

. . .and Vehq Scop -treated groups, n s 18 for the DHEASq Sal -treated group, and ns 32 for the DHEASq Scop -treated group. )P- . a aa . .))P-0.01 vs. the Vehq Sal -treated group; P-0.05, P-0.01 vs. the Vehq Scop -treated group Dunns test .

group. Only the latency remained significantly different

from the parameters showed by controls. These resultsindicated clearly that DHEAS allows a significant im-

provement 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 Vehq scopolamine -treated group, no signif-

.icant decrease was observed P)0.05, Fig. 4A . More-

over, the latencies measured in each trial, with an excep-

tion for trial 7, appeared significantly higher than th

showed by control animals. The PROG treatment, a .mgrkg Fig. 4A , did not affect the profiles showedcontrol or scopolamine-treated animals. The PROG

.Sal -treated group exhibited a significant decrease in la .cies P-0.01 , with no difference as compared with

.control group. The PROG q scopolamine -treated gr showed no significant decrease in latencies P)0.

with significant differences as compared with contr

particularly during the second acquisition day. The PR

treatment, at 20 mgrkg, affected moderately the profshowed by control or scopolamine-treated animals


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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 . .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

.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 .time spent within this area D were recorded. Veh: vehicle, sesame oil; Sal: saline; Scop: scopolamine. The numbers of animals per group were: n

. . . . . for the Vehq Sal -and Vehq Scop -treated groups, n s 15 for the PROG 2 q Sal -, PROG 2q Scop -, PROG 20q Sal - and PROG 20q Sc . a . .treated groups. )P-0.05, ))P-0.01 vs. the Vehq Sal -treated group; P-0.05 vs. the Vehq 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 PROGq scopolamine -treated

group showed a significant decrease in latencies P.0.01 , with significant differences as compared with

control group, particularly during the second acquisi


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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 scopolam . . .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

.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 . .the total time spent within this area D were recorded. The numbers of animals per group were: ns 18 for the Vehq Vehq Sal -treated group, n

. . .and Vehq Vehq Scop -treated group, n s 17 for the PROGq Veh q Sal -treated group, n s 18 for the PROG q Vehq Scop -treated group, n . . for the Vehq DHEASq Sal -treated group and n s 20 for the PROGq DHEASq Scop -treated group. )P-0.05, ))P-0.01 vs. the Vehq Ve

. a aa . o oo .Sal -treated group; P-0.05, P-0.01 vs. the Vehq Veh q Scop -treated group; P-0.05, P-0.01 vs. the Vehq DHEASq Scop -treated g .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 com-

pared 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 parame- .ters showed either by the control Vehq Sal -treated ani-

.mals or by the Veh q scopolamine -treated group duringthe 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 ob- .served, the control Veh

qVeh

qSal -treated group

showed a correct learning, since the latencies decreased . over training trials P-0.01, Fig. 5A . The Vehq Vehq

.scopolamine -treated group also showed some decrease in .latencies P-0.05, Fig. 5A . However, significant differ-

ences were observed as compared with controls, particu-

larly 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 . scopolamine-treated animals Fig. 5A . For the PROG

. VehqSal -treated group, the latencies decreased P. .0.01, Fig. 7A . The PROG qVehq scopolamine -trea

group showed no significant variation in latencies P.0.05, Fig. 5A , and the latencies measured from trials

8 appeared significantly higher than those measured control animals. For the Vehq DHEASq scopolami

treated group, the latencies decreased P-0.01, Fig. 5However, only the latency measured on trial 7 appea

significantly lower than the one measured for the scolamine-treated animals. For the PROG q DHEAS

.scopolamine -treated group, no decrease in latencies .observed P)0.05, Fig. 5B , the latencies appearing

nificantly higher compared with the control group f

trial 3 to 8, and on trial 4 and 7 compared with .Vehq DHEASqscopolamine -treated group.

The antagonist effect of PROG appeared clearly in

retention parameters, presented in the lower panel of F

5. The PROG treatment did not affect each paramete

compared with control or scopolamine-treated animHowever, the non-significant attenuation of latency . .5C and the significant increase in time Fig. 5D indu

by the DHEAS treatment were blocked by PROG, isignificant manner as compared with the Vehq DHE

. .qscopolamine -treated group P-0.01 each .

A similar antagonism of the DHEAS effect on

scopolamine-induced learning impairment could be

. Fig. 6. Antagonism by NE-100 of the neurosteroidal effects on the spontaneous alternation deficits induced by scopolamine in mice: A DHEAS . . .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 scopolam

.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. T . . .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

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 . a aa . .Vehq Veh q Sal -treated group; P-0.05, P-0.01 vs. the Vehq Vehq Scop -treated group; 8P-0.05 vs. the Vehq DHEASq Scop -tre

.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 scopolam . . .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

.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 . .the total time spent within this area D were recorded. The numbers of animals per group were: ns 19 for the Vehq Vehq Sal -treated group, n

. . . and Veh q Vehq Scop -treated group, n s 14 for the NE-100q Vehq Sal -treated group, and n s 15 for the NE-100q Vehq Scop -, Vehq DH. . . a aaq Sal - and NE-100q DHEASq Scop -treated groups. )P-0.05, ))P-0.01 vs. the Vehq Vehq Sal -treated group; P-0.05, P-0.0

. o oo . .the Veh q Vehq Scop -treated group; P-0.05, P-0.01 vs. the Vehq 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 receptor antagonist NE-100 was investigated1on 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 NE-

100 at 1 mgrkg andror DHEAS at 20 mgrkg. As already .observed, the control Veh

qVeh

qSal -treated group

showed a correct learning, with latencies decreasing signif- . icantly 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 ap-

peared 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 VehqSal -treated group, the latencies de- . creased P-0.01 similarly as for controls. The NE-100

.q Vehq scopolamine -treated group showed no signifi- .cant variation in latencies P)0.05 , and all latencies

appeared significantly higher than those measured for con-

.trol animals. For the Vehq DHEASq 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 .Vehq 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 scopolamine-

.treated animals Fig. 7C,D . However, the significant at-tenuation 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 neu-

rosteroids of the scopolamine-induced learning deficits in

mice using both a short-term memory test, the spontanew xalternation behavior in the Y-maze 1,2,36,37,54,63 , an

long-term memory test, place learning in a water-mw x w x39,69 . In accordance with previous studies 36,42 ,

alternation percentage in the Y-maze was decreased

scopolamine-treated animals from 70% to 50%, the cha

level, without any change in the number of arm entries

parallel, place learning in the water-maze was marke

affected. During acquisition sessions, the latencies spenfind the platform did not significantly decrease over tra

ing trials: the repeated measures ANOVA did not re

significance or the post-hoc comparison test did not sh

significant differences between trials. Furthermore, la

cies appeared significantly higher than the ones measu

for the control group. Then, during the retention sess

performed 48 h after the last training session, the param

ters showed by the scopolamine-treated group were

fected: a higher latency to reach the location of the p

form during training and a lower time spent within t

area were measured. Both tests thus allowed a quantita

measure of the scopolamine-induced learning deficits.None of the neurosteroids tested, in the 520 mgr

dose range, affected by itself the spontaneous alternatw xbehavior 35,38,41 or place learning in the water-m

.this study . Administration of DHEAS or PREGS

combination with scopolamine resulted in a dose-dep

dent attenuation of the scopolamine-induced deficits

both tests. In the Y-maze, the effects induced by the t

steroids appeared of similar extent, with a signific

attenuation observed at 20 mgrkg. PROG did not af

the scopolamine-induced alternation deficits, but beha

as an antagonist, by blocking the beneficial effects exer

by both neurosteroids in the Y-maze or by DHEAS in

water-maze. These results clearly confirmed the aamnesic effects of neurosteroids in an amnesia mo

involving blockade of the cholinergic muscarinic neu

transmission. The cognitive enhancing effects of neu

teroids in rodents have been previously reported u

several behavioral tests. Post-training central adminis

tion of DHEA or DHEAS improved the T-maze footsh

active avoidance or step-down type passive avoida

behaviors in mice, compared to the vehicle-treated animw x11,60 . In an extensive study, the same authors confirm

that i.c.v. administration of PREG, PREGS, DH

DHEAS, and testosterone, but not PROG, improved ac

w xavoidance retention in mice 10 . More recently, Frye w xSturgis 12 described the memory enhancing effects

PREG, PREGS and most efficiently DHEAS and 5a-pr

nan-3a-ol-20-one using female rats submitted to p

learning in the Morris water-maze test or to a dela

non-matching-to-sample test in a Y-maze. We did

observe any memory enhancing effects of neurosteroids

themselves in our tests, but the effects previously repor

could be observed in undertrained animals in the acw xavoidance test 10,11,60 or by using the Morris water-m

test, that allows the observation of learning improvem


11/14


w x12 . 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 im-

peded 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 donot cross the bloodbrain barrier at the same rate. Further-

more, sulfated steroids injected peripherally presumably do

not penetrate the brain, but may be converted to non-w xsulfated PREG of DHEA by microsomal sulfatase 17 .

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 sulfotrans-

ferases. 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 phar-

macological manipulations. DHEAS administered after

training at 20 mgrkg s.c. improved T-maze footshockw xactive avoidance in aged mice 9 . The DHEAS-treated 18-

and 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.w xFlood et al. 11 observed that DHEAS completely coun-

teracted the amnesia induced by anisomycin, an inhibitor

of protein synthesis, or scopolamine in mice submitted tow xthe footshock active avoidance test. Li et al. 21 reported

that peripheral administration of DHEAS alleviated thescopolamine-induced step-through type passive avoidance

deficits in mice. Furthermore, the steroid sulfatase in-

hibitor estrone-3-O-sulfamate administered alone enhanced

retention in the passive avoidance test and its administra-

tion in combination with DHEAS potentiated the neuros-w x w xteroidal effect 21 . Mayo et al. 44 reported that direct

infusions into the nucleus basalis magnocellularis of

PREGS or tetrahydroprogesterone enhanced or disrupted,

respectively, the performances of rats submitted to a two-

trial 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-in-duced 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 particu-

larly DHEAS and PREGS, potentiate the acetylcholine-de-

pendent memory processes. This effect was observed after

central or peripheral administration, and it could be ampli-

fied by the sulfatase inhibitor, demonstrating that the sul-w xfated form may be the most active one 12 . Recently,

w xRhodes et al. 58 provided a biochemical evidence for a

direct effect of neurosteroids on the cholinergic syste

DHEAS induced a dose-dependent increase in hippoc

pal acetylcholine release, measured in vivo using intrac

bral microdialysis in the anesthetized rat. Interestingly

the previously reported studies, DHEAS administered

ripherally showed its anti-amnesic effect at a similar o

mal dosage of 20 mgrkg, a dose that increased sign

cantly the hippocampal acetylcholine release in the st

w xby Rhodes et al. 58 .Interestingly, neurosteroids have also been reporte

alleviate the memory impairments induced by blockad

the NMDA-type of glutamatergic neurotransmiss

PREGS blocked the passive avoidance deficits induced the competitive NMDA receptor antagonists 3- "

. carboxypiperazin-4-yl -propyl-1-phosphonic acid C . w xand D-2-amino-5-phosphonovalerate D-AP5 26,27 . Th

w xCheney et al. 5 reported that PREGS, infused i.v

adrenalectomizedrcastrated rats, attenuated the amn

induced by the non-competitive NMDA receptor anta

nist dizocilpine, likely through an effect mediated by aw x

pregnanolone 5 . Finally, we reported that DHEAS, ministered systemically or centrally, attenuated the

zocilpine-induced deficits in spontaneous alternation

step-down type passive avoidance behaviors in mw x35,38 .

Different mechanisms can be proposed in order

explain the anti-amnesic effects of DHEAS or PREw xFlood et al. 10 proposed that the neurosteroid-indu

improvement of memory may imply their genomic effe

through the modulation of the rates and amounts of tr

scription of immediate-early genes. The immediate-e

genes induce a facilitation of translational processes le

ing to the synthesis of enzymes and proteins, some of th

being involved during the consolidation phase of the mory process. On the other hand, both DHEAS and PRE

are known to modulate negatively the GABA receAcomplex, potentiating the binding of GABAergic agon

w xand benzodiazepines 2325 . It is well-known that ben

diazepines are amnesic in rodents through their modula

of the GABA receptor, with GABAergic antagonAblocking and GABAergic agonists potentiating the m

w xory impairing effects of benzodiazepines 49,67 . Furtw xmore, Mayo et al. 43,44 reported that neurosteroids

fused into the nucleus basalis magnocellularis impro

the cognitive ability of rats in a two-trial recognition t

in the same manner as b-carbolines, which decreaseGABAergic neurotransmission. However, PREGS

peared active only after a post-training administration p

tocol, but not after a pre-training administration as w xserved for b-carbolines at low doses 43,44 . Neurostero

thus induce, through their negative modulation of GAB

receptors, a disinhibition of the cholinergic neurons of

nucleus basalis magnocellularis projecting into the h

pocampus, and concomittently an increase in excita

inputs through their positive modulation of NMDA recw xtors, mediating the memory enhancing effects 58 .


12/14


A third mechanism can be proposed from the present

study, through the neurosteroidsrs receptor interaction.1It 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 recep-1 . .tor agonists, q -N-allyl-normetazocine q -SKF-

. . . .10,047 , 1,3-di- 2-tolyl guanidine DTG , q -N-

cyclopropylmethyl-N-methyl-1,4-diphenyl-1-1-ethyl-but-3-

. w xen-1-ylamine hydrochloride JO-1784 3,4648 . In par-ticular, the steroidal effects were blocked by s receptor1

w antagonists, such as haloperidol, 1 2- 3,4-dichloro-. x .phenyl ethyl -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 receptor agonists. Consequently, we observed that in1the dizocilpine-induced amnesia model in mice the anti-

amnesic effect of DHEAS could be blocked by the s1 . receptor antagonist a- 4-fluorophenyl -4- 5-fluoro-2-

. . w xpyrimidinyl -1-piperazinebutanol BMY-14,802 38 , and

that PROG blocked the anti-amnesic effect of the selective . s receptor agonist 1- 3,4-dimethoxyphenethyl -4- 3-

1 . . w xphenyl propyl piperazine dihydrochloride SA4503 35 .

Moreover, s receptor agonists regulate the cholinergic1neurotransmission, as reported using in vitro or in vivo

w x .techniques. First, Siniscalchi et al. 65 reported that q -

SKF-10,047 potentiated the electrically evoked acetyl-

choline release from guinea-pig brain slices. Second,w x w xKobayhashi et al. 20 and Matsuno et al. 2830 demon-

.strated that several s receptor agonists, q -SKF-10,047,1 . ." -pentazocine, DTG, q -3-PPP, and SA4503 increased

the extracellular acetylcholine levels in the rat frontal

cortex and hippocampal formation, using in vivo micro-

dialysis in the rat. The increases were blocked by haloperi-

.dol and correlated with the binding affinities to the q -w3 xH SKF-10,047-labeled s sites, demonstrating the direct1

w xinvolvement of the s receptors 29 . Furthermore, s1 1receptor 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 administrationw x6,3133,64 . We observed here that the anti-amnesic ef-

fect induced by neurosteroids against the scopolamine-in-

duced learning impairment could be completely blocked

by the selective s receptor antagonist NE-100, demon-1strating a clear similar pharmacology between neuros-

teroids and s system on the acetylcholine-dependent1learning, as observed on the NMDA-dependent responses

and memory processes.

In summary, this study confirmed the anti-amnesic ef-

fects 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 receptor. Neurosteroids1may indeed induce their effects through different mecha-

nisms of action, involving rapid genomic events, disinhibi-

tion through their antagonism on GABA receptor com-A

plexes, and potentiation of excitatory inputs throug

facilitation of NMDA receptor activation and an ago

effect at s receptor. The differential involvement1these mechanisms in the cognitive effects of steroids m

depend on the physiologic steroidal levels, on the b

structure involved in the learning task, or on the admi

tration procedure. In particular, the consequences and th

apeutic opportunities of the interaction between neu

teroids and the s receptor in age-related and neurodeg1erative cognitive disabilities is currently under invest

tion.

Acknowledgements

Thanks are due to Jean Bayle for elaborating the ap

ratus used for behavioral testing, and to Taisho Pharmac .tical Tokyo, Japan for providing us NE-100. This w

was supported by INSERM.

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