BR A IN RE S E A RCH 1 1 65 ( 20 0 7 ) 2 1 –2 9

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Nanomolar concentrations of anabolic–androgenic steroids

amplify excitotoxic neuronal death in mixed
mouse cortical cultures

Rosamaria Orlando a,1 , Alessandra Caruso a,1 , Gemma Molinaro b , Marta Motolese a ,
Francesco Matrisciano a , Giuseppina Togna a , Daniela Melchiorri a ,
Ferdinando Nicoletti a,b , Valeria Bruno a,b,⁎
a
Department of Human Physiology and Pharmacology, University of Rome “La Sapienza”, Italy
b
I.N.M. Neuromed, Pozzilli, Italy

A R T I C LE I N FO AB S T R A C T

Article history: The use of anabolic–androgenic steroids (AASs) in the world of sport has raised a major
Accepted 20 June 2007 concern for the serious, sometimes life-threatening, side effects associated with these
Available online 10 July 2007 drugs. Most of the CNS effects are of psychiatric origin, and whether or not AASs are toxic to
neurons is yet unknown. We compared the effect of testosterone with that of the AASs, 19-
Keywords: nortestosterone (nandrolone), stanozolol, and gestrinone, on excitotoxic neuronal death
Excitotoxicity induced by N-methyl-D-aspartate (NMDA) in primary cultures of mouse cortical cells. In the
Androgen most relevant experiments, steroids were applied to the cultures once daily during the
Estrogen 4 days preceding the NMDA pulse. Under these conditions, testosterone amplified
Cortifical culture excitotoxic neuronal death only at very high concentrations (10 μM), whereas it was
Anabolic steroid protective at concentrations of 10 nM and inactive at intermediate concentrations. Low
concentrations of testosterone became neurotoxic in the presence of the aromatase
inhibitors, i.e. anastrozole and aminoglutethimide, suggesting that the intrinsic toxicity of
testosterone was counterbalanced by its aromatization into 17β-estradiol. As opposed to
testosterone, nortestosterone, stanozolol and gestrinone amplified NMDA toxicity at
nanomolar concentrations; their action was insensitive to aromatase inhibitors, but was
abrogated by the androgen receptor antagonist, flutamide. None of the AASs were toxic in
the absence of NMDA. These data suggest that AASs increase neuronal vulnerability to an
excitotoxic insult and may therefore facilitate neuronal death associated with acute or
chronic CNS disorders.
© 2007 Elsevier B.V. All rights reserved.

1. Introduction life-threatening, adverse effects that involve the liver, the

cardiovascular system, the male and female reproductive
The use of anabolic–androgenic steroids (AASs) for gains in systems, and the CNS (Ishak and Zimmerman, 1988; Ferenchick
strength and muscle mass is associated with serious, sometimes et al., 1992; Lukas, 1993; Rockhold, 1993; Trifunovic et al., 1995).

⁎ Corresponding author. Department of Human Physiology and Pharmacology, P.le Aldo Moro, 5, 00185 Rome, Italy. Fax: +39 0649912231.
E-mail address: valeria.bruno@uniroma1.it (V. Bruno).
1
Co-first authors.

0006-8993/$ ­ see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2007.06.047
22 BR A IN RE S EA RCH 1 1 65 ( 20 0 7 ) 2 1 –29
CNS adverse effects of AASs include stroke and psychiatric
symptoms, such as aggression, mania, psychosis, and suicide
attempts (Uzych, 1992; Thiblin et al., 1999; Hall et al., 2005;
Trenton and Currier, 2005). These effects might be related to the
AAS-induced decrease in plasma testosterone levels (Daly et al.,
2003). Testosterone and its metabolites, such as dihydrotestos-
terone and 17β-estradiol, are known to influence cognition,
anxiety and mood as a result of the activation of androgen
receptors (ARs) and estrogen β receptors (ERβs) (Edinger et al.,
2004; Beer et al., 2006; Edinger and Frye, 2005, 2006, 2007a,b). A
particular concern is raised by the suggestion that AASs may be
reinforcing independently of the awareness that they increase
muscle mass and physical performance, because they are
positive in the paradigms of self-administration and conditioned
place preference test in rats and hamsters (DiMeo and Wood,
2006; Ballard and Wood, 2005; Wood, 2004). The reinforcing
effects of AASs are prevented by naltrexone or dopamine
receptor antagonists, suggesting the involvement of opioidergic
and dopaminergic mechanisms (Peters and Wood, 2005; Wood,
2004). It is currently unknown whether AASs are toxic to neurons
and whether their abuse is a risk factor for acute or chronic
neurodegenerative disorders, although both neuroprotective and
neurotoxic effects has been described for testosterone. Genetic
alterations (i.e. a polyglutamine expansion) of ARs are associated
with progressive motor neuron disorders in the spinal and bulbar
muscular atrophy (SBMA or Kennedy's disease). Castration
restores function and neurofilament abnormalities of symptom-
atic males in a transgenic mouse model of SBMA (Chevalier-
Larsen et al., 2004), suggesting that androgens can be detrimental
to neurons under pathological conditions. Accordingly, testos-
terone depletion reduces ischemia–perfusion injury in rats
subjected to middle cerebral artery occlusion (Yang et al., 2002).
The few in vitro data available suggest that the influence of
androgens on neurodegeneration critically depends on the
treatment regimen, the type of insult, and the phenotype of cell
death. A single application of testosterone at supraphysiological
(micromolar) concentrations promotes apoptotic death in SH-
SY5Y neuroblastoma cells by altering the function of type-1
inositol-1,4,5-trisphosphate-receptor-mediated intracellular
Ca2+ signaling (Estrada et al., 2006). In contrast, cultured
cerebellar granule cells treated with 1 μM testosterone for 48 h
are less vulnerable to apoptotic death induced by oxidative stress
(Ahlbom et al., 2001), as are cultured cerebellar granule cells
prepared from 7-day-old rats treated 4 days before with a single
dose of testosterone propionate (Ahlbom et al., 1999). Testoster-
one, methyltestosterone, and the AAS mibolerone protect
cultured human neurons against apoptosis by serum deprivation
through the activation of ARs (Hammond et al., 2001). Surpris-
ingly, little is known on how androgens affect excitotoxic
neuronal death in spite of the large number of data obtained
with female sex steroids (see Discussion, and references therein).
Excitotoxicity refers to a particular mechanism of neuronal
death triggered by an excessive stimulation of glutamate
receptors (reviewed by Choi, 1994; Rothman and Olney, 1995).
This type of death, which may incorporate features of necrosis
and apoptosis, largely contributes to the pathophysiology of
major acute and chronic neurodegenerative disorders, includ-
ing stroke, brain traumatic injury, status epilepticus, hypogly-
cemic coma, Parkinson's disease, amyotrophic lateral sclerosis,
Huntington's disease, and Alzheimer's disease (reviewed by
Choi, 1988). In cultured HT-22 clonal mouse hippocampal cells,
high concentrations of testosterone (10 μM) amplify toxicity
induced by millimolar concentrations of glutamate (Yang et al.,
2002), whereas the androgen dehydroepiandrosterone (DHEA), a
metabolic precursor of testosterone, is protective (Cardounel
et al., 1999). However, toxicity by millimolar concentrations of
glutamate in HT-22 cells is not mediated by the stimulation of
glutamate receptors, but depends on the inhibition of cystine
uptake, which leads to a reduced synthesis of glutathione and
accumulation of reactive oxygen species (Tan et al., 1998). In in
vivo studies, androgenic neurosteroids (e.g. 5α-anstrostan-
3α,17β-diol), DHEA, testosterone, and the active testosterone
metabolite, dihydrotestosterone, are protective against hippo-
campal damage induced by the excitotoxins, kainic acid and
domoic acid (Frye and Reed, 1998; Cardounel et al., 1999; Azcoitia
et al., 2001; Ramsden et al., 2003). At least in one study, however,
neuroprotection was entirely dependent on the availability of
aromatase, the enzyme that converts testosterone into 17β-
estradiol (Azcoitia et al., 2001). Because most AASs are poor
substrates for aromatase, data obtained with testosterone in
vitro or in vivo are not predictive of how AASs affect excitotoxic
neuronal death.
Here, we have compared the effect of testosterone with that
of three synthetic AAS (nortestosterone, stanozolol, and gestri-
none) on excitotoxic neuronal death using as a model mixed
cultures of mouse cortical cells challenged with N-methyl-D-
aspartate (NMDA) (Rose et al., 1992). In the CNS, ARs are mainly
expressed in the cerebral cortex, mostly by neurons, with only
small subpopulations of androgen receptor-expressing astro-
cytes (DonCarlos et al., 2006). We report that AAS potently
amplify excitotoxic neuronal death through the activation of
ARs, and, as opposed to testosterone, their action is insensitive
to pharmacological inhibition of aromatase.
Fig. 1 – Excitotoxic neuronal death induced by increasing
concentrations of NMDA combined or not with MK-801 (1 μM)
in mixed cultures of mouse cortical cells. Neuronal death was
assessed by measuring LDH release (A) and incorporation of
Trypan blue (B) in the same cultures. Following Trypan blue
staining, dead neurons were counted in 3 random
microscopic fields/well. Values are means ± S.E.M. of 6
determinations from 2 independent experiments. *p < 0.05
(Student's t test) vs. the respective values obtained in the
absence of MK-801.
 BR A IN RE S E A RCH 1 1 65 ( 20 0 7 ) 2 1 –2 9 23

cultures preserve the physiological interplay between neurons

2. Results and astrocytes, and are widely used for the assessment of
excitotoxic neuronal death (Rose et al., 1992). Cultures were
We used mixed cultures of mouse cortical cells, in which challenged with NMDA for 10 min and excitotoxic neuronal
neurons were grown over a monolayer of astrocytes. These death was assessed 20–24 h later by combining Trypan blue

Fig. 2 – Modulation of NMDA toxicity by testosterone (A), nortestosterone (B), stanozolol (C), gestrinone (D), 17β-estradiol (E),
and progesterone (E) applied to the cultures once a day during the 4 days preceding a 10-min pulse with 60 μM NMDA. The
amount of neuronal death induced by 60 μM NMDA (from 39% to 51%) was set as 100% in all these experiments. Values are
expressed as percent of NMDA toxicity and represent the means ± S.E.M. of 6–12 determinations from 2 to 3 independent
experiments. *p < 0.05 one-way ANOVA + Fisher's PLSD) vs. NMDA alone. None of the steroids was toxic when applied for 4 days
at concentrations of 1 or 10 μM in cultures that were not exposed to NMDA (not shown).
24 BR A IN RE S EA RCH 1 1 65 ( 20 0 7 ) 2 1 –29

staining and LDH release. NMDA produced neuronal death in a

concentration-dependent fashion, with an apparent EC50
value of about 50–60 μM, and a maximal effect (85 ± 5% of
death) at concentrations N100 μM (Figs. 1A, B). As expected,
NMDA toxicity was abrogated by co-incubating the cultures
with the NMDA receptor antagonist, (SR, 10S)-(+)-5-methyl-
10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydro-
gen maleate (MK-801, 1 μM) (Figs. 1A, B). Using this paradigm
of “fast toxicity” (Rose et al., 1992), we examined the effect of a
4-day pretreatment with testosterone, 17β-estradiol, proges-
terone, and three AASs (stanozolol, nortestosterone, and
gestrinone). Cultures were challenged with a concentration
of NMDA that approximates the EC50 value (60 μM), and allows
to estimate both increases and reductions of excitotoxic death
reliably. Pretreatment with testosterone slightly reduced
NMDA toxicity at concentrations of 10 nM, was ineffective at
100 nM to 1 μM, but amplified toxicity at 10 μM (Fig. 2A). In
contrast, all three AASs enhanced NMDA toxicity at low
concentrations, with gestrinone behaving differently than
nortestosterone and stanozolol. Both nortestosterone and
stanozolol amplified excitotoxicity at concentrations N1 nM
(Figs. 2B, C). We could not determine the EC50 value of
nortestosterone because the enhancement of NMDA toxicity
did not reach plateau at the maximal concentration tested
(10 μM) (Fig. 2B). Stanozolol enhanced NMDA toxicity with an
apparent EC50 value of 10 nM and a maximal effect (50%
increase in NMDA toxicity) at 1–10 μM (Fig. 2C). Gestrinone was
already effective in enhancing NMDA toxicity at concentra-
tions as low as 0.1 nM. The action of gestrinone peaked at
100 nM (80% increase in NMDA toxicity) and then declined at
higher concentrations (Fig. 2D). None of the androgens we
have tested were toxic by themselves (i.e. in the absence of
NMDA) (not shown). A 4-day pretreatment with low concen-
trations (10 nM) of 17β-estradiol was substantially neuropro-
tective against NMDA toxicity. Neuroprotection was also
observed, albeit to a lesser extent, with 1 μM 17β-estradiol
(Fig. 2E). Progesterone was protective at all concentrations
tested, with a greater effect at 1 μM than at 10 or 100 nM
(Fig. 2E).
Because aromatase is expressed in neurons (reviewed by
Martini and Malcangi, 1991; Naftolin, 1994), a potential toxicity
of low concentrations of testosterone could have been
counterbalanced by the formation of 17β-estradiol during
the 4 days of incubation in culture. To examine this possibility,
testosterone was combined with the selective aromatase
inhibitor, anastrozole (25 nM) (Plourde et al., 1994; Geisler
et al., 1996), or with aminoglutethimide (10 μM), a drug that
inhibits multiple steps of steroid hormone biosynthesis,
including aromatization of testosterone into 17β-estradiol
(Cocconi, 1994). When combined with anastrozole or amino-
glutethimide, testosterone was no longer neuroprotective at
10 nM, and became neurotoxic at 1 μM (Figs. 3A, B). Fig. 3 – Modulation of NMDA toxicity by a 4-day pretreatment
Amplification of NMDA toxicity by 10 μM testosterone was with testosterone applied alone or combined with 25 nM
abrogated by the AR antagonist, flutamide (10 μM), which anastrozole (A), 10 μM aminoglutethimide (B) or 10 μM
instead did not affect neuroprotection by 10 nM testosterone flutamide (C). Toxicity was induced by 60 μM NMDA, and data
(Fig. 3C). Amplification of NMDA toxicity by nortestosterone, are expressed as indicated in Fig. 2. Values are means± S.E.M.
stanozolol, or gestrinone was prevented by flutamide, but was of 6 determinations from 2 independent experiments. p < 0.05
insensitive to anastrozole or aminoglutethimide (Figs. 4A–F). (one-way ANOVA + Fisher's PLSD) vs. NMDA alone (*) or vs.
Finally, we examined the effect of testosterone and the the corresponding values obtained in the absence of
three AASs applied in combination with NMDA during the anastrozole, aminoglutethimide or flutamide (#).
 BR A IN RE S E A RCH 1 1 65 ( 20 0 7 ) 2 1 –2 9 25

Fig. 4 – Modulation of NMDA toxicity by a 4-day pretreatment with nortestosterone (A, B), stanozolol (C, D) or gestrinone (E, F)
applied alone or combined with 25 nM anastrozole (A, C, E) aminoglutethimide (B, D, F) or flutamide (B, D, F). Toxicity was
induced by 60 μM NMDA, and data are expressed as indicated in Fig. 2. Values are means ± S.E.M. of 6 determinations from 2
independent experiments. p < 0.05 (one-way ANOVA + Fisher's PLSD) vs. NMDA alone (*) or vs. the corresponding values
obtained in the absence of aminoglutethimide or flutamide (#).
26 BR A IN RE S EA RCH 1 1 65 ( 20 0 7 ) 2 1 –29
Fig. 5 – Modulation of excitotoxic neuronal death by
testosterone (A), nortestosterone (B), and stanozolol, gestrinone,
17β-estradiol, and progesterone (C) applied during the 10-min
pulse (co-) with 60 μM NMDA. Values are means±S.E.M. of 6
determinations from 2 independent experiments. p <0.05
(one-way ANOVA+Fisher's PLSD) vs. NMDA alone (*) or vs. the
respective values with nortestosterone alone (#).
excitotoxic pulse, and then washed out 20 h prior to the
assessment of neuronal death. Using this particular protocol
of drug treatment, testosterone did not affect NMDA toxicity
(Fig. 5A), whereas nortestosterone amplified excitotoxic death
at concentrations N100 nM, and its action was prevented by
flutamide (Fig. 5B). Stanozolol amplified NMDA toxicity only at
10 μM, whereas gestrinone was toxic at 10 and 100 nM and
inactive at 10 μM (Fig. 5C). When combined with NMDA, 17β-
estradiol was protective at 10 nM but not at higher concentra-
tions, whereas progesterone was protective at all concentra-
tions tested (Fig. 5C).
In all experiments, we found neither incorporation of
Trypan blue nor apparent changes in morphology in the
astrocyte monolayer of the cultures when using NMDA alone,
AASs alone or NMDA in combination with AASs (not shown).
3. Discussion
We found that pretreatment with testosterone amplified
NMDA toxicity, but only at supraphysiological concentrations
(10 μM). Low concentrations of testosterone (10 nM), which
were otherwise neuroprotective, became neurotoxic in the
presence of the aromatase inhibitors, anastrozole or amino-
glutethimide. This indicates that testosterone is intrinsically
detrimental to cortical neurons challenged with NMDA, but
toxicity is counterbalanced by aromatization of testosterone
into 17β-estradiol. Estrogens have an established protective
activity in a variety in vitro and in vivo models of neuronal
death (reviewed by Inestrosa et al., 1998; Wise, 2002; McCul-
lough and Hurn, 2003; Marin et al., 2005; Hoffman et al., 2006;
Morale et al., 2006; Suzuki et al., 2006), and 17β-estradiol
attenuated excitotoxicity in our cultures. It is likely that the
low amounts of endogenous 17β-estradiol synthesized after
the addition of 10 nM testosterone were sufficient to afford
neuroprotection and counterbalanced the intrinsic toxicity of
testosterone. However, this putative endogenous neuropro-
tection was overwhelmed by increasing concentrations of
testosterone, which was in fact neurotoxic at 10 μM. Whether
this was due to saturation of aromatase activity or to
additional mechanisms that depend on the relative concen-
trations of 17β-estradiol and testosterone is unknown. Our
data are in agreement with the evidence that aromatase is
protective against hippocampal neuronal damage associated
with epilepsy (Zhou et al., 2007), and genetic deletion or
pharmacological inhibition of the enzyme amplify neuronal
death induced by the excitotoxins, kainic acid and domoic acid
(Azcoitia et al., 2001).
A 4-day pretreatment with nortestosterone, stanozolol and
gestrinone amplified excitotoxic death at low concentrations
(<1 μM), and their action was insensitive to anastrozole or
aminoglutethimide. This is consistent with the evidence that
nortestosterone is weakly aromatized (Dintinger et al., 1989),
whereas stanozolol and gestrinone are not substrates for
aromatase. Thus, the intrinsic toxicity of AASs was unopposed
by estrogen formation in our cultures. Nortestosterone
amplified NMDA toxicity at lower concentrations than stano-
zolol, which is consistent with its greater affinity at androgen
receptors (Roselli, 1998). Although nortestosterone can be
converted into the active androgen dihydronortestosterone by
 BR A IN RE S E A RCH 1 1 65 ( 20 0 7 ) 2 1 –2 9
5α-reductase, this process had little relevance in our cultures
if we take into account that nanomolar concentrations of
nortestosterone were more toxic than equimolar concentra-
tions of testosterone plus aminoglutethimide (compare
Figs. 3A and 4A) although dihydronortestosterone has a
lower affinity than dihydrotestosterone for androgen recep-
tors (Sundaram et al., 1995). Gestrinone amplified excitotoxic
death at extremely low concentrations (0.1 nM) but its action
declined at concentrations N100 nM. We have selected this
drug because it is structurally related to tetrahydrogestrinone
(THG), a highly potent AAS which is having a great impact in
the world of sport (Labrie et al., 2005). Gestrinone is an
anabolic androgen that behaves as a partial agonist of
progesterone receptors, is endowed with antiestrogenic activ-
ity, and is clinically useful in the treatment of endometriosis
(Rose et al., 1988; Peters, 1992). The lack of toxicity by high
concentrations of gestrinone might be ascribed to its proges-
terone-like activity, as suggested by the evidence that
progesterone itself was neuroprotective in our cultures.
A difference between the three AASs and testosterone
could also be observed when steroids when co-applied with
NMDA during the excitotoxic pulse. Under these conditions,
testosterone did not affect NMDA toxicity whereas nortestos-
terone, stanozolol and gestrinone retained their toxic activity,
albeit with a lower potency. This suggests that even a single
exposure to AASs may amplify excitotoxic death under
pathological conditions.
Taken together, our data offer the first evidence that AASs
are toxic to neurons that are challenged with an excitotoxic
insult. In principle, toxicity could result from a classic genomic
mechanism mediated by androgen receptors or, alternatively,
by a neurosteroid-like mechanism, which is independent of
androgen receptors. AASs may directly interact with GABAA
receptors by enhancing or reducing chloride currents depend-
ing on the brain region and the subunit composition of the
channel (Jorge-Rivera et al., 2000; Yang et al., 2005; Jones et al.,
2006). A direct inhibition of GABAA receptors might enhance
excitotoxicity by inducing membrane depolarization, thereby
relieving the Mg2+ blockade of the NMDA channel (Mayer et al.,
1984; Mayer and Westbrook, 1987). Androgens could also act
on NMDA receptors, either directly on the NR2B subunit,
which contains a steroid modulatory domain (Jang et al., 2004),
or indirectly on the sigma-1 receptor, which modulates NMDA
receptor complex (Yamamoto et al., 1995) and for which
testosterone and dehydroepiandrosterone show high affinity
(Debonnel et al., 1996; Monnet and Maurice, 2006). However, it
is unlikely that such mechanisms contribute to the enhance-
ment of NMDA toxicity in cultures exposed to AASs for the
following reasons: (i) AASs amplified neuronal death at
nanomolar concentrations, whereas the neurosteroid-like
action of these drugs at the GABAA receptor require micro-
molar concentrations (Yang et al., 2005; Jones et al., 2006);
(ii) the neuroprotective/neurotoxic effect of testosterone on
NMDA-induced neuronal degeneration required a 4-day
pretreatment, whereas a single application of AASs during
the NMDA pulse amplified excitotoxicity; and (iii) the action of
AASs was prevented by pharmacological blockade of ARs with
flutamide.
It cannot be excluded that androgens may affect neuronal
viability by acting through nongenomic mechanisms mediated
27
by the activation of ARs. It has been recently shown that
testosterone and dihydrotestosterone activate the mitogen-
activated protein kinase (MAPK) pathway in cultured hippo-
campal cells, which is related to their neuroprotective effects
against β-amyloid toxicity and depends on the presence of
ARs (Nguyen et al., 2005). Moreover, in C6 cells and in primary
cortical astrocytes, dihydrotestosterone increases the phos-
phorylation of both ERK and Akt, key effectors of the
neuroprotection-associated MAPK and phosphoinositide-3-
kinase pathways, an effect blocked by flutamide; however,
when a membrane-impermeable androgen is used, a suppres-
sion of ERK and Akt phosphorylation is observed, suggesting
that the activation of membrane-associated androgen recep-
tors may be detrimental to cells (Gatson et al., 2006; Gatson
and Singh, 2007).
Although in vivo data are still lacking, our evidence that
AASs enhance excitotoxic death with nanomolar potency
raises a serious concern for the use of these drugs for func-
tional enhancement or just for cosmesis, particularly because
abusers may have micromolar concentrations of AASs in their
brain (see Yang et al., 2005). Excitotoxic mechanisms can
be triggered by a series of vascular, metabolic and toxic
insults, as well as by an underlying neurodegenerative
disorder. Under these conditions, the abuse of AASs that are
not aromatizable into estrogens might accelerate the rate of
neuronal death.
4. Experimental procedures
4.1. Materials
Gestrinone was purchased from Cayman Chemical Company
(Ann Arbor, MI, USA). Anastrozole was kindly provided by Prof.
Adriana Maggi of the University of Milan. All other hormones
and chemicals were purchased from Sigma-Aldrich (Milan,
Italy). All hormones were dissolved in DMSO at the initial
concentration of 10 mM. The final concentration of DMSO
applied to the cultures was 0.1%.
4.2. Mixed cortical cultures
Mixed cortical cell cultures containing both neurons and
astrocytes were prepared from fetal mice at 14–16 days of
gestation, as described previously (Rose et al., 1992). Briefly,
dissociated cortical cells were plated in 15-mm multiwell
vessels (Falcon Primaria, Lincoln Park, NJ, USA) on a layer of
confluent astrocytes (7–14 days in vitro), using a plating
medium of MEM-Eagle's salts (supplied glutamine-free, and
lacking progesterone or other hormones) supplemented with
5% heat-inactivated horse serum, 5% fetal bovine serum,
glutamine (2 mM), and glucose (final concentration 21 mM).
Cultures were kept at 37 °C in a 5% CO2 humidified atmo-
sphere. After 3–5 days in vitro, non-neuronal cells division was
halted by 1–3 days of exposure to 10 μM cytosine arabinoside,
and cultures were shifted to a maintenance medium identical
to the plating medium but lacking fetal serum. Subsequent
partial medium replacement was carried out twice a week.
Only mature cultures (13–14 days in vitro) were used for the
experiments.
28 BR A IN RE S EA RCH 1 1 65 ( 20 0 7 ) 2 1 –29
4.3. Exposure to excitatory amino acids and AASs
Mixed cortical cell culture were exposed to 60 μM NMDA, at
room temperature in a HEPES-buffered salt solution contain-
ing (in mM): NaCl, 120; KCl, 5.4; MgCl2, 0.8; CaCl2, 1.8; HEPES, 20;
and glucose, 15 (and lacking hormones). Afterwards, cultures
were incubated at 37 °C for the following 20–24 h in MEM-
Eagle's supplemented with 15.8 mM NaHCO3 and glucose
<25 mM. Steroids were applied daily to the growing medium
during the 4 days preceding the NMDA pulse, in the presence
or absence of the aromatase inhibitors, anastrozole or
aminoglutethimide, and of the androgen receptor antagonist,
flutamide. Alternatively, steroids were applied in combination
with NMDA during the 10-min pulse; MK-801 (1 μM), when
used, was applied during the NMDA pulse.
4.4. Assessment of neuronal injury
Neuronal injury was measured in all experiments by exami-
nation of cultures with phase-contrast microscopy at 20×,
1 day after the insult, when the process of cell death was
largely complete. Neuronal damage was quantitatively
assessed by counting dead neurons stained with Trypan
blue. Stained neurons were counted from three random fields
per well. Neuronal damage was also assessed by measuring
LDH released in the medium using a commercial available kit
(Roche Diagnostic GmbH, Manneheim, Germany).
4.5. Statistics
Comparisons between experimental groups were made by
Student's t test and one-way ANOVA + Fisher's PLSD test.
Acknowledgments
This work was funded by a grant from the Italian Ministry of
Health, project 2006 on drugs used for doping in sport.
Anastrozole was kindly provided by Prof. Adriana Maggi,
Department of Pharmacology, University of Milan.
REFERENCES
Ahlbom, E., Grandison, L., Bonfoco, E., Zhivotovsky, B., Beccatelli,
S., 1999. Androgen treatment of neonatal rats decreases
susceptibility of cerebellar granule neurons to oxidative stress
in vitro. Eur. J. Neurosci. 11, 1285–1291.
Ahlbom, E., Prins, G.S., Ceccatelli, S., 2001. Testosterone protects
cerebellar granule cells from oxidative stress-induced cell
death through a receptor mediated mechanism. Brain Res. 892,
255–262.
Azcoitia, I., Sierra, A., Veiga, S., Honda, S., Harada, N., Garcia-Segura,
L.M., 2001. Brain aromatase is neuroprotective. J. Neurobiol. 47,
318–329.
Ballard, C.L., Wood, R.I., 2005. Intracerebroventricular
self-administration of commonly abused anabolic–androgenic
steroids in male hamsters (Mesocricetus auratus): nandrolone,
drostanolone, oxymetholone, and stanazolol. Behav. Neurosci.
119, 752–758.
Beer, T.M., Bland, L.B., Bussiere, J.R., Neiss, M.B., Wersinger, E.M.,
Garzotto, M., Ryan, C.W., Janowski, J.S., 2006. Testosterone loss
and estradiol administration modify memory in men. J. Urol.
175, 130–135.
Cardounel, A., Regelson, W., Kalimi, M., 1999.
Dehydroepiandrosterone protects hippocampal neurons
against neurotoxin-induced cell death: mechanism of action.
Proc. Soc. Exp. Biol. Med. 222, 145–149.
Chevalier-Larsen, E.S., O'Brien, C.J., Wang, H., Jenkins, S.C., Holder,
L., Lieberman, A.P., Merry, D.E., 2004. Castration restores
function and neurofilament alterations of aged symptomatic
males in a transgenic mouse model of spinal and bulbar
muscular atrophy. J. Neurosci. 24, 4778–4786.
Choi, D.W., 1988. Glutamate neurotoxicity and diseases of the
nervous system. Neuron 1, 623–634.
Choi, D.W., 1994. Calcium and excitotoxic neuronal injury. Ann. N.Y.
Acad. Sci. 747, 162–171.
Cocconi, G., 1994. First generation aromatase inhibitors:
aminoglutethimide and testololactone. Breast Cancer Res. Treat.
30, 57–80.
Daly, R.C., Su, T.P., Schmidt, P.J., Pagliaro, M., Pickar, D., Rubinow,
D.R., 2003. Neuroendocrine and behavioural effects of
high-dose anabolic steroid administration in male normal
volunteers. Psychoneuroendocrinology 28, 317–331.
Debonnel, G., Bergeron, R., de Montigny, C., 1996. Potentiation
by dehydroepiandrosterone of the neural response to
N-methyl-D-aspartate in the CA3 region of the rat dorsal
hippocampus : an effect mediated via sigma receptors.
J. Endocrinol. 150, S33–S42.
DiMeo, A.N., Wood, R.I., 2006. Self-administration of estrogen and
dihydrotestosterone in male hamsters. Horm. Behav. 49,
519–526.
Dintinger, T., Gaillard, J.L., Zwain, I., Bouhamidi, R., Silberzahn, P.,
1989. Synthesis and aromatization of 19-norandrogens in the
stallion testis. J. Steroid Biochem. 32, 537–544.
DonCarlos, L.L., Sarkey, S., Lorenz, B., Azcoitia, I., Garcia-Ovejero,
D., Huppenbauer, C., Garcia-Segura, L.M., 2006. Novel cellular
phenotypes and subcellular sites for androgen action in the
forebrain. Neuroscience 138, 801–807.
Edinger, K.L., Frye, C.A., 2005. Testosterone's anti-anxiety and
analgesic effects may be due in part to actions of its
5alpha-reduced metabolites in the hippocampus.
Psychoneuroendocrinology 30, 418–430.
Edinger, K.L., Frye, C.A., 2006. Intrahippocampal administration of
an androgen receptor antagonist, flutamide, can increase
anxiety-like behavior in intact and DHT-depleted male rats.
Horm. Behav. 50, 216–222.
Edinger, K.L., Frye, C.A., 2007a. Androgen's performance-enhancing
effects in the inhibitory avoidance and water maze tasks may
involve actions at intracellular androgen receptors in the dorsal
hippocampus. Neurobiol. Learn. Mem. 87, 201–208.
Edinger, K.L., Frye, C.A., 2007b. Androgen's effects to enhance
learning may be mediated in part through actions at estrogen
receptor-beta in the hippocampus. Neurobiol. Learn. Mem. 87,
78–85.
Edinger, K.L., Lee, B., Frye, C.A., 2004. Mnemonic effects of testosterone
and its 5alpha-reduced metabolites in the conditioned fear and
inhibitory avoidance tasks. Pharmacol. Biochem. Behav. 78,
559–568.
Estrada, M., Varshney, A., Ehrlich, B.E., 2006. Elevated testosterone
induces apoptosis in neuronal cells. J. Biol. Chem. 281,
24501–25492.
Ferenchick, G., Schwartz, D., Ball, M., Schwartz, K., 1992.
Androgenic–anabolic steroid abuse and platelet aggregation:
a pilot study in weight lifter. Am. J. Med. Sci. 303, 78–82.
Frye, C.A., Reed, T.A., 1998. Androgenic neurosteroids: anti-seizure
effects in an animal model of epilepsy.
Psychoneuroendocrinology 23, 385–399.
Gatson, J.W., Singh, M., 2007. Activation of a membrane-associated
androgen receptor promotes cell death in primary cortical
astrocytes. Endocrinology 148, 2458–2464.
 BR A IN RE S E A RCH 1 1 65 ( 20 0 7 ) 2 1 –2 9
Gatson, J.W., Kaur, P., Singh, M., 2006. Dihydrotestosterone
differentially modulates the mitogen-activated protein kinase
and the phosphoinositide-3-kinase/Akt pathways through the
nuclear and novel membrane androgen receptor in C6 cells.
Endocrinology 147, 2028–2034.
Geisler, J., King, N., Dowsett, M., Ottestad, L., Lundgren, S., Walton,
P., Kormeset, P.O., Lonning, P.E., 1996. Influence of anastrozole
(Arimidex), a selective, non-steroidal aromatase inhibitor, on
in vivo aromatization and plasma oestrogen levels in
postmenopausal women with breast cancer. Br. J. Cancer 74,
1286–1291.
Hall, R.C., Hall, R.C., Chapman, M.J., 2005. Psychiatric complications
of anabolic steroid abuse. Psychosomatics 46, 285–290.
Hammond, J., Le, Q., Goodyer, C., Gelfand, M., Trifiro, M., LeBlanc,
A., 2001. Testosterone-mediated neuroprotection through the
androgen receptor in human primary neurons. J. Neurochem.
77, 1319–1326.
Hoffman, G.E., Merchenthaler, I., Zup, S.L., 2006. Neuroprotection
by ovarian hormones in animal models of neurological disease.
Endocrine 29, 217–231.
Inestrosa, N.C., Marzolo, M.P., Bonnefont, A.B., 1998. Cellular and
molecular basis of estrogen's neuroprotection. Potential relevance
for Alzheimer's disease. Mol. Neurobiol. 17, 73–86.
Ishak, K.G., Zimmerman, H.J., 1988. Drug-induced and toxic
granulomatous hepatitis. Bailliere's clin. gastroenterol. 2,
463–480.
Jang, M.K., Mierke, D.F., Russek, S.J., Farb, D.H., 2004. A steroid
modulatory domain on NR2B controls N-methyl-D-aspartate
receptor proton sensitivity. Proc. Natl. Acad. Sci. U. S. A. 101,
8198–8203.
Jones, B.L., Whiting, P.J., Henderson, L.P., 2006. Mechanisms of
anabolic androgenic steroid inhibition of mammalian
epsilon-subunit-containing GABAA receptors. J. Physiol. 573,
571–593.
Jorge-Rivera, J.C., McIntyre, K.L., Henderson, L.P., 2000. Anabolic
steroids induce region- and subunit-specific rapid modulation
of GABA(A) receptor-mediated currents in the rat forebrain.
J. Neurophysiol. 83, 3299–3309.
Labrie, F., Luu-The, V., Calvo, E., Martel, C., Cloutier, J., Gauthier, S.,
Belleau, P., Morissette, J., Levesque, M.H., Labrie, C., 2005.
Tetrahydrogestrinone induces a genomic signature typical of a
potent anabolic steroid. J. Endocrinol. 184, 427–433.
Lukas, S.E., 1993. Current perspectives on anabolic–androgenic
steroid abuse. Trends Pharmacol. Sci. 14, 61–68.
Marin, R., Guerra, B., Alonso, R., Ramirez, C.M., Diaz, M., 2005.
Estrogen activates classical and alternative mechanisms to
orchestrate neuroprotection. Curr. Neurovasc. Res. 2, 287–301.
Martini, L., Malcangi, R.C., 1991. Androgen metabolism in the
brain. J. Steroid Biochem. Mol. Biol. 39, 819–828.
Mayer, M.L., Westbrook, G.L., 1987. Permeation and block of
N-methyl-d-aspartic acid receptor channels by divalent
cations in mouse cultured central neurones. J. Physiol. 394,
501–527.
Mayer, M.L., Westbrook, G.L., Guthrie, P.B., 1984.
Voltage-dependent block by Mg2+ of NMDA responses in
spinal cord neurons. Nature 309, 261–263.
McCullough, L.D., Hurn, P.D., 2003. Estrogen and ischemic
neuroprotection: an integrated view. Trends Endocrinol.
Metab. 14, 228–235.
Monnet, P.P., Maurice, T., 2006. The sigma protein 1 as a target for the
non-genomic effect of neuro(active) steroids : molecular,
physiological and behavioural aspects. J. Pharm. Sci. 100, 93–118.
Morale, M.C., Serra, P.A., L'Episcopo, F., Tirolo, C., Caniglia, S.,
Testa, N., Gennuso, F., Giaquinta, G., Rocchitta, G., Desole, M.S.,
Miele, E., Marchetti, B., 2006. Estrogen, neuroinflammation and
neuroprotection in Parkinson's disease: glia dictates resistance
versus vulnerability to neurodegeneration. Neuroscience 138,
869–878.
29
Naftolin, F., 1994. Brain aromatization of androgens. J. Reprod.
Med. 39, 257–261.
Nguyen, T.V., Yao, M., Pike, C.J., 2005. Androgens activate
mitogen-activated protein kinase signaling: role in
neuroprotection. J. Neurochem. 94, 1639–1651.
Peters, F., 1992. Multicentre study of gestrinone in cyclical breast
pain. Lancet 339, 205–208.
Peters, K.D., Wood, R.I., 2005. Androgen dependence in hamsters:
overdose, tolerance, and potential opioidergic mechanisms.
Neuroscience 130, 971–981.
Plourde, P.V., Dyroff, M., Dukes, M., 1994. Arimidex: a potent and
selective fourth-generation aromatase inhibitor. Breast Cancer
Res. Treat. 30, 103–111.
Ramsden, M., Shin, T.M., Pike, C.J., 2003. Androgens modulate
neuronal vulnerability to kainate lesion. Neuroscience 122,
573–578.
Rockhold, R.W., 1993. Cardiovascular toxicity of anabolic steroids.
Annu. Rev. Pharmacol. Toxicol. 33, 497–520.
Rose, G.L., Dowsett, M., Mudge, J.E., White, J.O., Jeffcoate, S.L., 1988.
The inhibitory effects of danazol, danazol metabolites, gestrinone,
and testosterone on the growth of human endometrial cells in
vitro. Fertil. Steril. 49, 224–228.
Rose, K., Goldberg, M.P., Choi, D.W., 1992. Cytotoxicity in murine
neocortical cell cultures. In: Tyson, C.A., Frazier, J.M. (Eds.),
Methods in Toxicology, vol. 1. San Diego Academics, San Diego,
pp. 46–60.
Roselli, C.E., 1998. The effect of anabolic–androgenic steroids on
aromatase activity and androgen receptor binding in the rat
preoptic area. Brain Res. 792, 271–276.
Rothman, S.M., Olney, J.W., 1995. Excitotoxicity and the NMDA
receptor – still lethal after eight years. Trends Neurosci. 18,
57–58.
Sundaram, K., Kumar, N., Monder, C., Bardin, C.W., 1995. Different
patterns of metabolism determine the relative anabolic activity
of 19-norandrogens. J. Steroid Biochem. Mol. Biol. 53, 253–257.
Suzuki, S., Brown, C.M., Wise, P.M., 2006. Mechanisms of
neuroprotection by estrogen. Endocrine 29, 209–215.
Tan, S., Wood, M., Maher, P., 1998. Oxidative stress induces a form of
programmed cell death with characteristics of both apoptosis
and necrosis in neuronal cells. J. Neurochem. 71, 95–105.
Thiblin, I., Runeson, B., Rajs, J., 1999. Anabolic androgenic steroids
and suicide. Ann. Clin. Psychiatry 11, 223–231.
Trenton, A.J., Currier, G.W., 2005. Behavioural manifestations of
anabolic steroid use. CNS Drugs 19, 571–595.
Trifunovic, B., Norton, G.R., Duffield, M.J., Avraam, P., Woodiwiss,
A.J., 1995. An androgenic steroid decreases left ventricular
compliance in rats. Am. J. Physiol. 268, H1096–H1105.
Uzych, L., 1992. Anabolic–androgenic steroids and psychiatric-related
effects: a review. Can. J. Psychiatry 37, 23–28.
Wise, P.M., 2002. Estrogens and neuroprotection. Trends
Endocrinol. Metab. 13, 229–230.
Wood, R.I., 2004. Reinforcing aspects of androgens. Physiol. Behav.
83, 279–289.
Yamamoto, H., Yamamoto, T., Sagi, N., Klenerová, V., Goji, K.,
Kawai, N., Baba, A., Takamori, E., Moroji, T., 1995. Sigma ligands
indirectly modulate the NMDA receptor-ion channel complex
on intact neuronal cells via sigma 1 site. J. Neurosci. 15, 731–736.
Yang, S.H., Perez, E., Cutright, J., Liu, R., He, Z., Day, A.L.,
Simpkins, J.W., 2002. Testosterone increases neurotoxicity of
glutamate in vitro and ischemia–reperfusion injury in an
animal model. J. Appl. Physiol. 92, 195–201.
Yang, P., Jones, B.L., Henderson, L.P., 2005. Role of the alpha
subunit in the modulation of GABA(A) receptors by anabolic
androgenic steroids. Neuropharmacology 49, 300–316.
Zhou, L., Lehan, N., Wehrenberg, U., Disteldorf, E., von Lossow, R.,
Mares, U., Jarry, H., Rune, G.M., 2007. Neuroprotection by
estradiol: a role of aromatase against spine synapse loss after
blockade of GABA(A) receptors. Exp. Neurol. 203, 72–81.