The effects of DHEA, 3β-hydroxy-5α-androstane-6,17-dione, and 7-amino-DHEA analogues on short term and long term memory in the mouse

Article abstract of DOI:10.1016/j.steroids.2009.06.010

Neurosteroids have been reported to modulate memory processes in rodents. Three analogues of dehydroepiandrosterone (DHEA), two of them previously described (7β-aminoDHEA and 7β-amino-17-ethylenedioxy-DHEA), and a new one (3β-hydroxy-5α-androstane-6,17-dione) were synthesized, and their effects were evaluated on memory. This study examined their effects on long term and short term memory in male (6 weeks old) NMRI mice in comparison with the reference drug. Long term memory was assessed using the passive avoidance task and short term memory (spatial working memory) using the spontaneous alternation task in a Y maze. Moreover, the effects of DHEA and its analogues on spontaneous locomotion were measured. In all tests, DHEA and analogues were injected at three equimolar doses (0.300-1.350-6.075 μM/kg). DHEA and its three analogues administered immediately post-training at the highest doses (6.075 μM/kg, s.c.) improved retention in passive avoidance test. Without effect per se in the spatial working memory task, the four compounds failed to reverse scopolamine (1 mg/kg, i.p.)-induced deficit in spontaneous alternation. These data suggested an action of DHEA and analogues in consolidation of long term memory particularly when emotional components are implied. Moreover, data indicated that pharmacological modulation of DHEA as performed in this study provides derivatives giving the same mnemonic profile than reference molecule.

Full text of DOI:10.1016/j.steroids.2009.06.010

Steroids 74 (2009) 931–937
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
The effects of DHEA, 3␤-hydroxy-5␣-androstane-6,17-dione, and 7-amino-DHEA
analogues on short term and long term memory in the mouse
Marc-Antoine Bazina,1, Laïla El Kihela,∗, Michel Boulouardb, Valentine Bouëtb, Sylvain Raulta
a Centre d’Etudes et de Recherche sur le Médicament de Normandie, UFR des Sciences Pharmaceutiques, Boulevard Becquerel, 14032 Caen cedex, France
b Groupe Mémoire et Plasticité comportementale, UFR des Sciences Pharmaceutiques, Université de Caen Basse–Normandie, 5 rue Vaubénard, 14032 Caen cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Neurosteroids have been reported to modulate memory processes in rodents. Three analogues of
dehydroepiandrosterone (DHEA), two of them previously described (7␤-aminoDHEA and 7␤-amino-17-
ethylenedioxy-DHEA), and a new one (3␤-hydroxy-5␣-androstane-6,17-dione) were synthesized, and
their effects were evaluated on memory. This study examined their effects on long term and short term
memory in male (6 weeks old) NMRI mice in comparison with the reference drug. Long term memory
was assessed using the passive avoidance task and short term memory (spatial working memory) using
the spontaneous alternation task in a Y maze. Moreover, the effects of DHEA and its analogues on spon-
taneous locomotion were measured. In all tests, DHEA and analogues were injected at three equimolar
doses (0.300–1.350–6.075 ␮M/kg). DHEA and its three analogues administered immediately post-training
at the highest doses (6.075 ␮M/kg, s.c.) improved retention in passive avoidance test. Without effect per
se in the spatial working memory task, the four compounds failed to reverse scopolamine (1 mg/kg, i.p.)-
induced deficit in spontaneous alternation. These data suggested an action of DHEA and analogues in
consolidation of long term memory particularly when emotional components are implied. Moreover,
data indicated that pharmacological modulation of DHEA as performed in this study provides derivatives
giving the same mnemonic profile than reference molecule.
Received 23 January 2009
Received in revised form 24 June 2009
Accepted 25 June 2009
Available online 3 July 2009
Keywords:
DHEA analogues
Long term memory
Short term memory
Scopolamine
Spontaneous activity
Neurosteroids
Published by Elsevier Inc.
1. Introduction
DHEA and DHEAS exhibit a broad spectrum of biological actions,
from interactions with brain development to complex processes
Steroid hormones exert important functions in the control of
growth, maturation and differentiation of the central and periph-
eral nervous systems. These actions have long been attributed
exclusively to steroid hormones secreted by endocrine glands, i.e.
adrenal, ovary, and testis. However, during the last decade, it has
been shown that nerve cells (both neurons and glial cells) are capa-
ble of synthesizing bioactive steroids, now called neurosteroids
[1]. Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone
sulfate (DHEAS) were the first steroids identified in large concen-
trations in the rat brain [2]. P450c17, the enzyme that is required
for synthesis of DHEA from pregnenolone, is found in specific neu-
rons of embryonic rodent brains [3]. DHEA and DHEAS were the
most abundant neurosteroids which were both synthesized and
accumulated in the nervous system to levels at least in part inde-
pendent of peripheral steroidogenesis [4]. It has been reported that
such as learning, enhancing memory and cognitive performances
during development and into adulthood [5–10]. Vallée et al. [11]
have reviewed the role of dehydroepiandrosterone and its sulfate
ester on learning and memory in cognitive aging. DHEA and DHEAS
increase the effects of the excitatory neurotransmitter glutamate
[12], and decrease the effects of the inhibitory neurotransmitter
GABA [13].
DHEA and DHEAS also have neuroprotective effects. They protect
hippocampal neurons against glutamate excitotoxicity [14]. Other
authors showed that neuroprotection by DHEA, but not DHEAS, was
mediated through inhibition of nitric oxide (NO) production and
inhibition of calcium-sensitive NO synthase (NOS) activity, caused
by NMDA stimulation [15].
Neurosteroids, which are involved in the regulation of stress
responses, anxiety, sleep, neurodegenerative processes, aggressive
behavior, and cognitive activities, were considered as key factors of
chemical neurotransmission [16].
In addition, many studies showed clearly the beneficial role of
DHEA administration on Alzheimer’s disease [17], immune system
[18], aging [19], obesity [20], diabetes [21], cardiovascular disease
[22], cancer [23–24], metabolic changes [25], acquired immune
deficiency syndrome [26], and depression [27]. Svec and Porter have
∗
Corresponding author. Tel.: +33 2 31 56 68 12; fax: +33 2 31 56 68 03.
E-mail address: laila.elkihel@unicaen.fr (L.El. Kihel).
Current address: Université de Nantes, Nantes Atlantique Universités, IICiMed
1
EA1155, UFR des Sciences Pharmaceutiques, Laboratoire de Chimie Thérapeutique,
1 rue Gaston Veil, BP 53508, F-44000 Nantes, France.
0039-128X/$ – see front matter. Published by Elsevier Inc.
doi:10.1016/j.steroids.2009.06.010

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M.-A. Bazin et al. / Steroids 74 (2009) 931–937
Fig. 1. DHEA and DHEA analogues.
reviewed the actions of exogenous DHEA in animal experimental
models and in humans [28]. Although DHEA is purported to have
many beneficial effects, there is only limited evidence to support its
use in humans and there have been few clinical trials that clearly
substantiate the efficacy and safety for use of DHEA supplements.
It has been well established that a significant portion of circu-
lating DHEA is further metabolized to its 7-oxygenated derivatives
(7-oxo-DHEA, 7␣- and 7␤-hydroxy-DHEA) in several different tis-
sues, including liver and brain [29–31]. These DHEA metabolites
are widely studied and seem to have even a wider activity than the
parent steroids [32–35]. However, it is not well clarified what are
the physiological functions of DHEA and its metabolites. Moreover,
the broad effects of DHEA limit its therapeutic use. On the other
hand, few DHEA analogues have been developed to our knowledge.
Several monohydroxylated derivatives of DHEA (4␣, 5␣, 7␣, 7␤,
11␤ and 16␣) and androst-5-ene-3␤,17␤-diol have been synthe-
sized and evaluated for the induction of the thermogenic enzymes.
Only 7-oxygenated derivatives induced thermogenic enzymes [36].
Other authors have demonstrated that the reduction of the C-
5–C-6 double bond of DHEA, C-7 hydroxylation or oxidation of
DHEA (such as DHEA metabolites) reduces the derivatives’ ability
to activate NMDA receptors. The hydroxylation on C-17 position
of pregnenolone also reduces this ability [37]. Since DHEA has a
broad spectrum of activities, chemical modification on C-7, C-17,
C-5, and/or C-6 position of the steroid core might lead to changes
related to one of these activities.
furic acid/ethanol (2:8) on TLC and subsequent heating. Column
chromatography was carried out using silica gel 60 (0.063–0.2 mm)
(Merck). Melting points were determined on a Kofler block. IR spec-
tra were recorded on a PerkinElmer BX FT-IR spectrometer. EI mass
spectra were recorded on a Jeol-GCmate (GC–MS system) spec-
trometer with ionisation energy from 30 to 40 eV. 1H NMR and 13
C
NMR spectra were recorded using CDCl3 respectively at 400 MHz
(Jeol Lambda 400 spectrometer) and at 100 MHz. Chemical shifts
are reported relative to TMS; J values are given in Hz. 13C NMR spec-
tra are 1H-decoupled. Elemental analyses were performed at the
“Institut de Recherche en Chimie Organique Fine” (Rouen, France).
2.1.2. Chemical synthesis
2.1.2.1. 7ˇ-Amino-17,17-ethylenedioxy-androst-5-en-3ˇ-ol (I). Co-
mpound I has been prepared according to a reported procedure
[44].
2.1.2.2. 7ˇ-Amino-3ˇ-hydroxy-androst-5-en-17-one
(II). Com-
pound II has been prepared according to a reported procedure
[44].
2.1.2.3. 3ˇ-Acetoxy-androst-5-en-17-one (1). Acetic anhydride
(26 mL, 277 mmol) was added dropwise to a solution of dehy-
droepiandrosterone (10 g, 35 mmol) in pyridine (25 mL). The
solution was stirred at room temperature and under argon for 12 h.
Ice water (20 mL) was poured into the mixture. The white precip-
itate formed was dissolved in CH2Cl2 (200 mL). The organic layer
was washed successively with 1 M HCl (3× 20 mL), 5% NaHCO3 (1×
30 mL), brine (1× 30 mL) and water (1× 30 mL), dried over Na2SO4
and evaporated under reduced pressure. The crude product was
recrystallized from acetone to give 3␤-acetoxy-androst-5-en-17-
one (1) (10.9 g, 95%) as a white powder [44].
In this pathway, and in our studies related to the synthesis of
amino and polyaminosteroids [38–44], we now report the synthe-
sis of 3␤-hydroxy-5␣-androstane-6,17-dione and we examine its
effect and that of two other DHEA analogues on memory in mice
(Fig. 1). With this goal, we have first tested the capacity of new
derivatives to improve retention of passive avoidance (long term
memory), and second, if they would reverse amnesia induced by
scopolamine in spontaneous alternation test (short term memory).
2.1.2.4. 3ˇ-Acetoxy-6-nitro-androst-5-en-17-one (2). Fuming nitric
acid (7.7 mL, 158.6 mmol) was added dropwise to a solution of 3␤-
acetoxy-androst-5-en-17-one (1) (10.0 g, 30.3 mmol) in acetic acid
(25 mL). The solution was stirred at room temperature under room
atmosphere for 2 h. The precipitate formed was dissolved in CH2Cl2
(300 mL). The organic layer was washed successively with 0.5N
NaOH (2× 100 mL), brine (1× 50 mL) and water (1× 50 mL), dried
over Na2SO4 and evaporated under reduced pressure. The crude
product was recrystallized from diethyl ether/ethyl acetate (10:1)
to give 3␤-acetoxy-6-nitro-androst-5-en-17-one (2) (9.4 g, 83%) as
a white powder. Mp 219 ◦C. IR (KBr) ꢀ (cm−1): 2940–2864 (C–H
alkane), 1736 (C O ester), 1725 (C O ketone), 1516, 1364, 1245
2. Experimental
2.1. Chemistry
2.1.1. General remarks
All solvents were distilled and dried prior to use. Reagents and
materials were obtained from commercial suppliers and were used
without further purification. The reactions were monitored by TLC
on Kieselgel-G (Merck Si 254 F) layers (0.25 mm thick). The spots
were detected using a UV lamp (254 nm) and by spraying with sul-

M.-A. Bazin et al. / Steroids 74 (2009) 931–937
933
(N–O), 1029. 1H NMR (CDCl3): 0.63 (s, 3H, Me18 ), 1.13 (s, 3H, Me19),
2.02 (s, 3H, –OCOCH3), 4.61–4.70 (m, 1H, H3). 13C NMR (CDCl3): 13.7
(C18), 19.9 (C19), 20.5 (–OCOCH3), 21.4, 21.9, 27.1, 31.2, 31.3, 31.4,
32.4, 35.8, 36.3, 38.1, 47.5, 49.2, 51.3, 71.9 (C3), 138.0, 146.1, 170.2
(–OCOCH3), 219.8 (C17). MS (EI, 30 eV) m/z (%): 315 (6) [M+•−AcOH],
300 (9) [M+•−(AcOH+Me•)], 286 (100), 271 (49), 258 (24), 231 (24),
163 (19). Anal. calcd. for C21H29NO5: C, 67.18; H, 7.79; N, 3.73, found:
C, 66.84; H, 7.51; N, 3.43.
dimethylsulfoxide (DMSO, Sigma–Aldrich) and then in saline solu-
tion, final vehicle being 5% DMSO in saline. All drugs were
administered in a volume less than 0.1 mL/10 g body weight.
In all tests, DHEA and analogues I–III were s.c. injected at
3 equimolar doses: 0.300–1.350–6.075 ␮mol/kg. In the sponta-
neous alternation task, scopolamine and DHEA/derivatives were
all administered 30 min before the test; DHEA/derivatives were
injected sub-cutaneously and scopolamine (1 mg/kg) was injected
intra-peritoneally immediately after. The drug doses and adminis-
tration routes were selected according to previous studies [35,45].
For clarification, the terms D1, D2 and D3 were attributed to the
three increasing doses: 0.300, 1.350 and 6.075 ␮mol/kg, respec-
tively in the results part (corresponding table and figures).
2.1.2.5. 3ˇ-Acetoxy-5˛-androstane-6,17-dione (3). A suspension of
zinc dust (22.3 g, 341.3 mmol) in acetic acid (150 mL) was refluxed
for 20 min. After cooling to room temperature, 3␤-acetoxy-6-nitro-
androst-5-en-17-one (2) (8.0 g, 21.3 mmol) dissolved in acetic acid
(15 mL) was added. The mixture was heated at 80 ◦C for 6 h. Zinc
was filtered and the solution was diluted with water and extracted
with CH2Cl2 (4× 100 mL) and the organic layer was washed with
aqueous 0.5N NaOH (2× 50 mL) and then with water (1× 100 mL),
dried over MgSO4 and CaCl2, and evaporated under reduced pres-
sure. The crude product was purified by column chromatography
(silica gel, cyclohexane/ethyl acetate (9:1)) to give 3␤-acetoxy-5␣-
androstane-6,17-dione (3)(6.3 g, 86%) as awhitepowder. Mp 188 ◦C.
IR (KBr) ꢀ (cm−1): 2938–2860 (C–H alkane), 1734 (3× C O), 1698,
1242, 1033. 1H NMR (CDCl3): 0.81 (s, 3H, Me18 ), 0.88 (s, 3H, Me19),
2.04 (s, 3H, –OCOCH3), 4.64–4.73 (m, 1H, H3). 13C NMR (CDCl3): 13.0
(C18), 13.8 (C19), 20.7 (–OCOCH3), 21.3, 21.6, 26.1, 26.8, 31.1, 35.6,
36.3, 37.4, 40.9, 45.3, 48.1, 51.6, 53.9, 56.5, 72.6, 170.6 (–OCOCH3),
209.3 (C6), 219.8 (C17). MS (EI, 30 eV) m/z (%): 346 (1) [M+•], 286
(2) [M+•−AcOH], 271 (1) [M+•−(AcOH+Me•)], 117 (2), 87 (10), 85
(66), 83 (100). Anal. calcd. for C21H30O4: C, 72.80; H, 8.73, found: C,
73.02; H, 8.68.
2.2.2.1. Locomotor activity. Spontaneous activity was recorded with
a photoelectric activity meter [46] (APELAB) composed with Plex-
iglas enclosures (25.5 cm × 20.5 cm × 9 cm) equipped with two
crisscross photo-cells in a dark enclosure. The number of interrup-
tions of the photoelectric beams by each animal (n = 10 per group)
was counted during a 5 min test 24 h after treatments.
2.2.2.2. Long term memory. A step-through type passive avoidance
box (LETICA LE 872), divided into a wide, white and illuminated
compartment (22 cm × 21 cm × 30 cm) and a small, black and dark
compartment (7.3 cm × 7.5 cm × 14 cm) with a grid floor delivering
electric foot shock, was used [47,48]. A guillotine door separated
the two compartments. During a first training trial (“Session 1”),
each mouse (n = 13 per group) was placed in the white compart-
ment. As soon as the mouse had entered into the dark compartment
(the step-through maximal latency was fixed at 50 s), the door was
closed and the mouse received a unique inescapable electric shock
(0.4 mA, 2 s). Twenty-four hours later, the mouse was tested for
retention (“Session 2”); mouse was placed again into the white
compartment and the time until it re-entered into the dark com-
partment was measured (the step-through maximal latency was
fixed at 300 s; no electric shock was delivered). Drug was adminis-
tered just after the session 1.
2.1.2.6. 3ˇ-Hydroxy-5˛-androstane-6,17-dione (III). Aqueous 10%
KOH(30 mL)was added toasolutionof3␤-acetoxy-5␣-androstane-
6,17-dione (3) (5.0 g, 14.4 mmol) in ethanol (20 mL) and the mixture
was then refluxed for 4 h. Ethanol was evaporated under reduced
pressure and the aqueous solution was acidified with 2N HCl (pH 4).
The solution was extracted with ethyl acetate (2× 100 mL) and the
organic layer was washed with water (1× 50 mL), dried over MgSO4
and evaporated under reduced pressure. The crude product was
purified by column chromatography (silica gel, cyclohexane/ethyl
acetate (7:3)) to give 3␤-hydroxy-5␣-androstane-6,17-dione (III)
(1.4 g, 32%) as a pale yellow powder. Mp 184 ◦C. IR (KBr) ꢀ (cm−1):
2.2.2.3. Short term memory. Immediate spatial working memory
performances were assessed by recording spontaneous alternation
behavior in a single-session Y-maze test [49,50]. The maze con-
sisted of three equally spaced arms (22 cm long, 6.5 cm wide, walls
of 10 cm high) made of black-painted wood. Thirty minutes after
vehicle or drug injection, each mouse (n = 10 per group) was placed
at the end of one arm and allowed to explore the maze freely during
a 5 min session. The number and the sequence of arm entries were
recorded by the observer. An arm entry was scored when all four
feet crossed into the arm. Alternation behavior is defined as con-
secutive entries into all three arms. The percentage of alternation
was calculated as a memory index by the (number of alterna-
tion/maximal theoretical number of alternation) × 100. DHEA and
derivatives I–III were administered 30 min before the test. In the
case of co-administration with scopolamine, neurosteroid deriva-
tives were administered just after scopolamine which was injected
30 min before the test.
3420 (br, O–H), 2943–2857 (C–H alkane), 1735 and 1706 (2× C
O
ketone), 1452, 1376, 1261, 1058. 1H NMR (CDCl3): 0.79 (s, 3H, Me18 ),
0.87 (s, 3H, Me19), 3.53–3.62 (m, 1H, H3). 13C NMR (CDCl3): 13.0
(C19), 13.6 (C18), 20.6, 21.5, 29.7, 30.4, 31.0, 35.5, 36.5, 37.2, 40.7,
45.2, 48.0, 51.5, 53.7, 56.7, 70.1 (C3), 210.0 (C6), 219.9 (C17). MS (EI,
30 eV) m/z (%): 304 (100) [M+•], 289 (25) [M+•−Me•], 248 (29), 233
(21), 139 (89), 95 (77). Anal. calcd. for C19H28O3: C, 74.96; H, 9.27,
found: C, 75.12; H, 9.45.
2.2. Behavioral study
2.2.1. Animals
Experiments were performed on male NMRI mice (6 weeks old)
obtained from Centre d’Elevage René Janvier (Le Genest, France).
They were maintained in a regulated environment (22 2 ◦C;
55 10% humidity) under a reversed 12–12 h light/dark cycle (light
on between 20:00 and 8:00) with food and water available ad libi-
tum. All experiments were complied with the European Directives
and the French law on animal experimentation.
2.2.2.4. Statistical analyses. All experimental series were analyzed
separately as independent series with their own control groups.
In all studies, results were expressed as means SD. p-Values less
than 0.05 were considered to be significant. For the spontaneous
alternation and locomotor activity experiments, data were ana-
lyzed through a one-way analysis of variance (ANOVA) with “dose
of drug” (for the dose–response studies) or “pharmacological treat-
ment” (for the combined treatment studies) as an independent
factor, followed, when appropriate, by a post hoc multiple com-
parison test (PLSD of Fischer) to locate the principal effect. Passive
2.2.2. Drug administration
Scopolamine hydrochloride, from Sigma–Aldrich (Lyon, France)
was dissolved in physiological saline (1 mg/kg) before use and
prepared daily. DHEA and derivatives I–III were solubilized in

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M.-A. Bazin et al. / Steroids 74 (2009) 931–937
Scheme 1. Synthesis of 3␤-hydroxy-5␣-androstane-6,17-dione (III). Reagents and reaction conditions: (i) Ac2O, Py, r.t., 12 h; (ii) HNO3, AcOH, r.t., 2 h; (iii) Zn, AcOH, 80 ◦C,
6 h; (iv) aqueous 10% KOH, EtOH, reflux, 4 h.
Table 1
Spontaneous locomotor activity expressed as the mean ( SD) number of beam interruptions in the activity box over a single 5 min session in mice 24 h after administration
of either DMSO (5% in saline: control) or DHEA, I, II, and III at three different doses (D1, D2, and D3, see details in Section 2). ANOVA analysis did not reveal any effect of the
four drugs (n = 10 in each group).
Control: 116.8 60.6
Control: 132.0 67.6
Control: 107.0 47.7
Control: 97.5 47.4
DHEA D1: 139.4 52.4
DHEA D2: 114.6 55.6
DHEA D3: 110.9 54.9
I D1: 147.3 79.4
I D2: 121.8 43.0
I D3: 140.9 30.2
II D1: 94.5 57.7
II D2: 110.4 51.1
II D3: 102.6 56.4
III D1: 104.4 57.7
III D2: 82.9 60.1
III D3: 74.9 52.3
avoidance learning was analyzed by a repeated measure ANOVA
with interdependent “session” effect and an independent “group”
effect. In the case of a significant principal effect and an interaction,
a one-way ANOVA was undertaken and followed, if appropriate, by
a PLSD of Fischer to locate the principal effect. Statistical analysis
was performed with STATVIEW® software.
3␤-Hydroxy-5␣-androstane-6,17-dione (III) was prepared as
shown in Scheme 1. 3␤-Acetoxy-androst-5-en-17-one (1) was
treated with high density nitric acid (d = 1.51) in anhydrous acetic
acid to give the 6-nitro derivative 2 in 83% yield. The nitro deriva-
tive 2 was then treated with zinc in acetic acid to give ketone 3. The
latter ketone was saponified by ethanolic potassium hydroxide to
give the corresponding deacetylated ketone III.
3. Results
3.2. Behavioral studies in mice
3.1. Chemical study
3.2.1. Locomotor activity
7␤-Amino-17,17-ethylenedioxy-androst-5-en-3␤-ol (I) and 7␤-
amino-3␤-hydroxy-androst-5-en-17-one (II) have been prepared
according to a reported procedure [44].
Results concerning horizontal locomotor activities registered
24 h after s.c. injection of DHEA and compounds I–III are indicated
in Table 1. None of these derivatives altered spontaneous activ-
Fig. 2. Passive avoidance performances in control, DHEA, I, II, and III groups expressed by the step-through latency in seconds (mean SD). Each drug was administrated at
three different doses (D1, D2, and D3, see details in Section 2). All the four drugs increased the step-through latency at the retrieval session at the highest doses. #Indicates a
significant difference with control group; *indicates differences with the two doses D1 and D2.

M.-A. Bazin et al. / Steroids 74 (2009) 931–937
935
Fig. 3. Spontaneous alternation performances (mean percentages SD). (A) Effect of administration of either saline (control) or DHEA, I, II, and III at three different doses
(D1, D2, and D3, see details in Section 2) on spontaneous alternation percentages in the Y-maze in mice. ANOVA did not reveal any effect of the four drugs. (B) Effect of
scopolamine and DHEA, I, II, and III at three different doses on spontaneous alternation. ANOVA.
ity [ANOVA F(3.36) = 0.529; p = 0.6651 for DHEA; F(3.36) = 0.814,
p = 0.49 for I; F(3.36) = 0.165, p = 0.49 for II, F(3.36) = 0.0.606, p = 0.62
for III)]. These data suggest that like DHEA, its derivatives also have
no effect on spontaneous locomotor activity.
the DHEA injected groups, only DHEA D3 differed from control
(p < 0.0001); the same holds true for the mice injected with I, II,
or III, in which the highest doses induced an increase in the latency
to enter the dark compartment compared to controls (p < 0.0001
for the three synthetic DHEA analogues at the dose D3). Accord-
ingly, for each drug the dose D3 significantly differed from D1 and
D2 (p < 0.05). These results suggest that at the maximal dose tested
(6.075 ␮mol/kg) the four derivatives increase the retention perfor-
mances of passive avoidance.
3.2.2. Long term memory (passive avoidance test)
At the acquisition session, there was no difference between
groups; all mice entered the dark compartment within 50 s. At
the retention session, there was a global difference between
groups (ANOVA F(12.56) = 9.105; p < 0.0001), a global difference
between the sessions showing that the retention session was signif-
icantly different from the acquisition session in all groups (ANOVA
F(1.12) = 1310.4; p < 0.0001) and an interaction group × session
(ANOVA F(12.1) = 9.241; p < 0.0001). Fisher’s PLSD revealed that
the highest doses (D3) of the three drugs induced an increase in
step-through latency compared to controls (see Fig. 2). Indeed, in
3.2.3. Short term memory
Working memory was assessed through spontaneous alter-
nation in the Y-maze. The results (Fig. 3A) did not reveal any
deleterious effect of the drugs, neither for the DHEA itself nor for
its analogues, administered alone. Indeed, mice treated with the
three different doses of DHEA or with the three analogues displayed

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M.-A. Bazin et al. / Steroids 74 (2009) 931–937
similar spontaneous alternation performances as controls (about
70%) and this was not modified whatsoever by the doses adminis-
tered [ANOVA F(3.36) = 1.921; p = 0.1437 for DHEA; F(3.36) = 0.256,
p = 0.8564 for I; F(3.36) = 0.666, p = 0.5782 for II, F(3.36) = 0.569,
p = 0.6389 for III]. Fig. 3B shows the results obtained in condi-
tion of scopolamine-induced amnesia. Scopolamine was efficient
in all experiments to induce deficits in spontaneous alternation
shifting spontaneous alternation percentages from 70% to 50%.
Indeed in all tested groups, there was a global group effect [ANOVA
F(4.45) = 5.072, p = 0.0018 for DHEA + scopolamine; F(4.45) = 3.820,
p = 0.0093 for I + scopolamine; F(4.45) = 7.056, p = 0.0002 for II;
F(4.45) = 7.306, p = 0.0001 for III + scopolamine]. The difference
was significant between control and scopolamine groups but not
between mice administered by scopolamine and animals treated
with DHEA or DHEA analogues [Fischer’s PLSD: control vs. scopo-
lamine: p < 0.05 (in all experiments); DHEA (D1, D2, D3) vs.
scopolamine: p > 0.05; I (D1, D2, D3) vs. scopolamine: p > 0.05; II
(D1, D2, D3) vs. scopolamine: p > 0.05; III (D1, D2, D3) vs. scopo-
lamine: p > 0.05]. These results suggest that DHEA or its analogues
were not able to reverse the deficits induced by scopolamine at any
dose tested.
and possibly its derivatives tested here could improve memory per-
formances through an action potentiating glutamate transmission.
Moreover, the effects of neurosteroids in the passive avoidance
task seemed memory specific since in the present study, at the
active dose none of these compounds altered spontaneous locomo-
tor activity measured 24 h after s.c. injection. In addition, the active
dose (20 mg/kg for DHEA) is the same that reported as improving
retention deficit of spatial reference memory in aging mice [35]. In
the spontaneous alternation task, the four neurosteroids, without
per se effects on immediate working memory performances, failed
to improve scopolamine-induced memory deficit. These results
(especially, those concerning DHEA) did not fully agree with those
of Urani et al. [57] who demonstrated that DHEA sulfate at 20 mg/kg
(but not at 5 and 10 mg/kg) produced a significant attenuation of
scopolamine (2 mg/kg, s.c.)-induced alternation deficit. Such dis-
crepancies could be attributable to differences in experimental
conditions (strain: Swiss mice in Urani et al. study, NMRI in our case;
light/dark cycle: light on at 8 h for Urani et al., light on at 20 h for us;
dimensions of the Y maze and protocol used: maze exploration dur-
ing 8 min for Urani et al., 5 min herein). Concerning comparison of
potential differences in mnemonic effects of the four compounds,
these results confirm that they had similar profiles: compounds
I–III as well as DHEA improved consolidation of long term mem-
ory but failed to improve scopolamine-induced working memory
deficit. Moreover, these data suggested a particular action of DHEA
in emotionally loaded memory [52]. In conclusion, these results
seemed particularly innovative because the new DHEA analogues
synthesized and tested are not biosynthetic products of DHEA.
4. Discussion
4.1. Chemical study
In a previous paper [44] we described the synthesis of
7␤-amino-17,17-ethylenedioxy-androst-5-en-3␤-ol (I), 7␤-amino-
3␤-hydroxy-androst-5-en-17-one (II) and 7␣-amino-3␤-hydroxy-
androst-5-en-17-one. In that study, we only tested the ␤ epimer
(II), which was synthesized in greatest yield.
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