REVIEW ARTICLE

Neurosteroid Biosynthesis Regulates Sexually Dimorphic Fearand Aggressive Behavior in Mice

Graziano Pinna Æ Roberto Carlos Agis-Balboa Æ Fabio Pibiri Æ Marianela Nelson ÆAlessandro Guidotti Æ Erminio Costa

Accepted: 14 April 2008 / Published online: 13 May 2008

� Springer Science+Business Media, LLC 2008

Abstract The neurosteroid allopregnanolone is a potent

positive allosteric modulator of GABA action at GABAA

receptors. Allopregnanolone is synthesized in the brain

from progesterone by the sequential action of 5a-reductase

type I (5a-RI) and 3a-hydroxysteroid dehydrogenase

(3a-HSD). 5a-RI and 3a-HSD are co-expressed in cortical,

hippocampal, and olfactory bulb glutamatergic neurons and

in output neurons of the amygdala, thalamus, cerebellum,

and striatum. Neither 5a-RI nor 3a-HSD mRNAs is

expressed in glial cells or in cortical or hippocampal

GABAergic interneurons. It is likely that allopregnanolone

synthesized in principal output neurons locally modulates

GABAA receptor function by reaching GABAA receptor

intracellular sites through lateral membrane diffusion.

This review will focus on the behavioral effects of

allopregnanolone on mouse models that are related to a

sexually dimorphic regulation of brain allopregnanolone

biosynthesis. Animal models of psychiatric disorders,

including socially isolated male mice or mice that receive a

long-term treatment with anabolic androgenic steroids

(AAS), show abnormal behaviors such as altered fear

responses and aggression. In these animal models, the

cortico-limbic mRNA expression of 5a-RI is regulated in a

sexually dimorphic manner. Hence, in selected glutama-

tergic pyramidal neurons of the cortex, CA3, and

basolateral amygdala and in granular cells of the dentate

gyrus, mRNA expression of 5a-RI is decreased, which

results in a downregulation of allopregnanolone content. In

contrast, 5a-RI mRNA expression fails to change in the

striatum medium spiny neurons and in the reticular tha-

lamic nucleus neurons, which are GABAergic.

By manipulating allopregnanolone levels in glutamater-

gic cortico-limbic neurons in opposite directions to improve

[using the potent selective brain steroidogenic stimulant

(SBSS) S-norfluoxetine] or induce (using the potent 5a-RI

inhibitor SKF 105,111) behavioral deficits, respectively, we

have established the fundamental role of cortico-limbic

allopregnanolone levels in the sexually dimorphic regulation

of aggression and fear. By selectively targeting allopreg-

nanolone downregulation in glutamatergic cortico-limbic

neurons, i.e., by improving the response of GABAA recep-

tors to GABA, new therapeutics would offer appropriate and

safe management of psychiatric conditions, including

impulsive aggression, irritability, irrational fear, anxiety,

posttraumatic stress disorders, and depression.

Keywords Allopregnanolone � 5a-reductase type I �Anabolic androgenic steroids (AAS) �Selective brain steroidogenic stimulants (SBSSs) �Social isolation � Posttraumatic stress disorders (PTSD)

Introduction

Men and women differ in temperament and personality

traits. Men are much more likely to be involved in acts of

violence, violent crime, domestic violence, and sexual

assault [1]. They commit 80% of all violent crimes and

99% of rapes [1]. The sex hormone testosterone has been

considered a trigger for these behavioral differences but the

molecular mechanisms and neural circuits involved remain

unclear [2].

Special issue article in honor of Dr. Ji-Sheng Han.

G. Pinna (&) � R. C. Agis-Balboa � F. Pibiri � M. Nelson �A. Guidotti � E. Costa

Psychiatric Institute, Department of Psychiatry, College of

Medicine, University of Illinois at Chicago, 1601W, Taylor

Street, Chicago, IL 60612, USA

e-mail: gpinna@psych.uic.edu

123

Neurochem Res (2008) 33:1990–2007

DOI 10.1007/s11064-008-9718-5

Prenatal exposure to high testosterone levels during

embryonic development may lead to complex changes in

neuroendocrine function that may impact adult cognitive

function [3, 4]. Studies in twins have suggested that a female

twin who has been prenatally exposed to testosterone pro-

duced by her male counterpart expresses a masculinization

of personality traits, including increased aggression [5, 6]. In

laboratory mammals, fetuses located between two males are

exposed to higher levels of testosterone and they express

greater aggressiveness than those situated between two

female fetuses [5]. In female rodents, testosterone promotes

the onset of stereotypical male behaviors, likely following a

synaptic remodeling of target cortico-limbic circuits [2, 7].

In humans, recent advances in functional brain imaging

have identified critical neural circuits in cortico-limbic

structures involved in the modulation of fear responses,

aggressiveness, anxiety, and sexual behaviors that appear

to be affected in mood disorders [8]. These circuits include

the amygdala, hippocampus, and the medial prefrontal

cortex (mPFC) [8, 9]. In rodents, the olfactory system has

also been implicated [10].

In humans, negative emotions can be controlled by

neurons located in basolateral amygdaloid nuclei [11, 12];

in addition, a downregulation of the mPFC-mediated inhi-

bition of amygdala function results in a slower extinction of

aversive responses, leading to an increased expression of

impulsivity and aggression [8, 12]. A hyperactive amyg-

dala, secondary to abnormal regulation by the mPFC,

facilitates aggressiveness [13]. Decreasing amygdalar

hyperactivity may be a primary effect of antiaggressive

agents, allowing increased neurotransmission between the

amygdala and several cortical areas [14, 15]. Hence, one

may infer that an imbalance in the cortico-limbic circuit

provides a neural substrate favoring impulsive aggression,

altering sexual behavior, forming emotional memories, and

facilitating memory retrieval [16].

Involved in these synaptic connections are excitatory

glutamatergic projecting neurons that express GABAA

receptors in their dendritic shafts and that therefore are

responsive to GABAergic mechanisms by pharmacological

interventions [17]. It is noteworthy that these cortical

glutamatergic neurons include pyramidal neurons charac-

terized by an intense production of the GABAA receptor-

active neurosteroid allopregnanolone [18].

This review focuses on the behavioral effects of allo-

pregnanolone biosynthesis downregulation induced in mice

either by social isolation or by anabolic androgenic steroid

(AAS) [e.g., testosterone propionate (TP)] administration.

By manipulating allopregnanolone levels in cortico-limbic

structures in opposite directions, we have established the

fundamental role of the changes in cortico-limbic allo-

pregnanolone levels in the regulation of aggression and fear

responses [19–22].

Neurosteroid Biosynthesis

The term ‘‘neurosteroid’’ [23, 24] does not refer to a

particular chemical class of steroids but indicates that their

synthesis and action site is the CNS. Progesterone, for

example, which is a hormone produced and secreted in the

circulatory system by both the ovaries and the adrenal

glands, is considered a neurosteroid when it is synthesized

and released in the brain or in peripheral nerves. Allo-

pregnanolone is the most abundant and efficacious

endogenous positive allosteric modulator of the action of

GABA at GABAA receptors and maintains the local levels

of GABAergic neurotransmission efficacy at the required

neurophysiological level [25–27].

Allopregnanolone is synthesized from progesterone in

the brain by the sequential action of two enzymes: (i)

5a-reductase type I (5a-RI), which reduces progesterone to

5a-dihydroprogesterone (5a-DHP) and is the rate-limiting

step enzyme in allopregnanolone biosynthesis [28]; and (ii)

3a-hydroxysteroid dehydrogenase (3a-HSD), which con-

verts 5a-DHP into allopregnanolone by a reductive

reaction or converts allopregnanolone into 5a-DHP via an

oxidative reaction (Fig. 1) [29]. When present in a nmolar

concentration range, allopregnanolone not only potentiates

the inhibitory signals mediated by the release of GABA but

also increases the efficacy of muscimol or that of other

positive allosteric modulators of GABA action at GABAA

receptors, including benzodiazepines and barbiturates

[25–27, 29–31].

The positive allosteric modulation of GABA action at

GABAA receptors elicited by allopregnanolone can be

documented in vivo and in vitro either by administering

this neurosteroid and documenting its behavioral action or

by correlating brain region-specific changes in neurosteroid

biosynthesis rates and recording consequent changes in

GABAA receptor function [25–28].

For this purpose, our laboratory has developed a highly

specific gas-chromatographic-mass-spectrometric (GC-MS)

method that allows accurate measurement in the attomolar

range of specific neurosteroids [25, 32, 33]. Using this

method, one can measure the neurosteroid content of discrete

brain areas in specific brain nuclei and even in laser micro-

dissected neuronal populations [25, 26, 32–34].

Local Neurosteroid Bioavailability to Cortico-Limbic

Circuitry Neurons

Independent from the neurotransmitter chemical phenotype

(e.g., glutamate or GABA), the neurosteroidogenic

enzymes 5a-RI and 3a-HSD are highly expressed in brain

primary output neurons (e.g., cortico and hippocampal

pyramidal neurons, granular cells, reticulo-thalamic

Neurochem Res (2008) 33:1990–2007 1991

123

neurons, medium spiny striatum and nucleus accumbens

neurons, and Purkinje neurons) and are virtually absent in

GABAergic cortical interneurons and glial cells [18].

GABAergic neurons of the reticular thalamic nucleus

express high levels of 5a-RI and 3a-HSD and their nerve

endings may secrete allopregnanolone and release GABA

in the proximity of postsynaptic GABAA receptors located

on the dendrites and somata of glutamatergic thalamocor-

tical output neurons [35]. Similar considerations are also

expected for allopregnanolone synthesized by medium

spiny GABAergic neurons in the caudate or putamen and

also very likely by Purkinje cells that modulate GABAA

receptors expressed postsynaptically on cell bodies or

dendrites of deep cerebellar nuclei neurons.

Alternatively, allopregnanolone synthesized in principal

cortico-limbic glutamatergic output neurons may act: (1) in

a paracrine fashion at GABAA receptors located on cell

bodies or dendrites of distal cortical pyramidal neurons

(Fig. 2, arrow 1); (2) in an autocrine fashion at GABAA

receptors located on dendrites or cell bodies of cortical

pyramidal neurons after being secreted from dendrites or

cell bodies of the same neurons [18] (Fig. 2, arrow 2); and

(3) allopregnanolone might not be released but may access

GABAA receptors located on the cell bodies or dendritic

arborization of glutamatergic neurons, acting at the intra-

cellular sites of the GABAA receptors by lateral diffusion

into plasma membranes [18, 36, 37] (Fig. 2, arrow 3).

Several psychiatric conditions, including anxiety,

impulsive aggression, premenstrual dysphoria, and post-

traumatic stress disorders, and various animal experiments

modeling these psychiatric conditions [i.e., environmental

factor—(e.g., social isolation) or pharmacological manip-

ulation-induced (e.g., TP treatment)], have been associated

with a decrease of allopregnanolone availability in cortico-

limbic neuronal circuits [21, 38–41].

Based on the knowledge acquired with the cellular

characterization of enzymes operative in neurosteroid

synthesis and metabolism, one can hypothesize that these

behavioral alterations could be elicited at least in part by a

local GABAA receptor neurotransmission downregulation

mediated by a decrease in allopregnanolone steady-state

levels in one or more synaptic stations of the cortico-limbic

circuit complex.

Testosterone

↓ ↓

SKF _ ? SNFLX

Pregnenolone Progesterone5α-RI

Allopregnanolone

Cholesterol

P450scc

5α−DHP

3α-HSD3β-HSD

+

Testosterone

Fig. 1 In the brain, allopregnanolone is synthesized from progester-

one by the sequential action of: (i) 5a-reductase type I (5a-RI), which

reduces progesterone to 5a-dihydroprogesterone (5a-DHP) and func-

tions as the rate-limiting step enzyme in allopregnanolone

biosynthesis; and (ii) 3a-hydroxysteroid dehydrogenase (3a-HSD),

which either converts 5a-DHP into allopregnanolone (reductive

reaction) or allopregnanolone into 5a-DHP (oxidative reaction).

17b-(N,N-diisopropylcarbamoyl)-androstan-3,5-diene-3-carboxylic

acid (SKF 105,111, SKF) is a potent competitive 5a-RI inhibitor [32].

S-norfluoxetine stimulates the accumulation of allopregnanolone by

targeting 3a-HSD [44]. In cortico-limbic structures, testosterone has

been proposed to inhibit 5a-RI expression [52, 96] but the molecular

mechanism is unknown. P450 scc, P450 cholesterol side-chain

cleavage; 3b-HSD, 3b-hydroxysteroid dehydrogenase; SNFLX,

S-norfluoxetine

Fig. 2 A diagrammatic representation of local allopregnanolone

biosynthesis and action on GABAA receptors located on synaptic

membranes of cortical pyramidal neurons. GABA released from

GABAergic interneurons activates a family of postsynaptic and

extrasynaptic GABAA receptors. Allopregnanolone facilitates the

synaptic inhibitory action of GABA at postsynaptic and extrasynaptic

GABAA receptors by a paracrine (arrow 1) or autocrine (arrow 2)

mechanism or may access GABAA receptors by acting at the

intracellular sites (arrow 3) of the GABAA receptors. (Modified from

Herd et al. [31]). GAD, glutamic acid decarboxylase; GABA,

c-aminobutyric acid; StAR, steroidogenic acute regulatory protein;

MBR, mitochondria benzodiazepine receptor; DBI, diazepam binding

inhibitor; P450 scc, P450 cholesterol side-chain cleavage; 3a-HSD,

3a-hydroxysteroid dehydrogenase; 5a-RI, 5a-reductase type I;

3b-HSD, 3b-hydroxysteroid dehydrogenase

1992 Neurochem Res (2008) 33:1990–2007

123

The purpose of this review is not only to apply this

knowledge in the elucidation of the neurochemical mech-

anisms and neuronal circuitry operative in the sexual

dimorphic regulation of mouse aggression, but also to

discuss new therapeutic avenues to treat neuropathophysi-

ologies related to GABAergic dysfunction in psychiatric

disorders.

The Role of Allopregnanolone in the Regulation

of GABAergic Neurotransmission and Behavioral

Abnormalities

Several lines of evidence support the concept that a

downregulation of brain allopregnanolone biosynthesis

plays a role in the development and maintenance of

GABAA receptor neurotransmission deficits leading to

abnormal emotional behaviors, including impulsive

aggression, posttraumatic stress disorders, anxiety, and

depression [21, 39, 40, 42].

It is well established that protracted social isolation in

rodents affects behavioral, physiological, and biochemical

parameters including susceptibility to GABAergic drugs

[25, 29, 38, 43]. Behaviorally, socially isolated mice

become emotionally unstable, showing (a) a time-dependent

aggressiveness toward an intruder (Fig. 3); (b) increased

locomotor activity when exposed to a novel environment;

and (c) increased intensity of contextual fear and anxiety

[19, 21, 22]. In socially isolated mice, these behavioral

abnormalities are associated with a 5a-RI expression

downregulation and reduction of allopregnanolone levels

without any decrease in the expression of 3a-HSD (19, 28;

Fig. 3). The brain structures that have been reported to be

dysfunctional and thus are considered responsible for con-

sequent emotional disorders are primarily the olfactory

bulb, mPFC, hippocampus, and amygdala [19, 21, 22, 41].

Systemic administration of non-sedative doses of allopreg-

nanolone that normalize the social isolation-induced

downregulation of cortico-limbic allopregnanolone content

abolishes the behavioral abnormalities observed in socially

isolated male mice [19, 21, 22]. Moreover, the selective

serotonin reuptake inhibitors (SSRIs) S-fluoxetine or

S-norfluoxetine isomers normalize the downregulation of

brain allopregnanolone content and block the expression of

aggression, anxiety, and fear responses [19, 21, 22]. The S

isomers of fluoxetine and norfluoxetine with a higher

potency than the R isomers normalize the decrease of pen-

tobarbital-induced sedation in isolated male mice at doses

that are virtually devoid of 5-HT reuptake inhibitory activity

[20, 21]. Incidentally, reuptake of 5-HT is not stereoselec-

tive [20]. The mechanisms by which fluoxetine and

norfluoxetine and other SSRIs cause a rapid increase of

brain allopregnanolone levels in socially isolated mice

remain to be clarified. In vitro studies reported by Griffin

and Mellon [44] showed that fluoxetine and various other

SSRIs fail to affect recombinant 5a-RI activity in concen-

trations as high as 50 lM, whereas in a lM concentration

range, these drugs enhance the ability of 3a-HSD to convert

5a-DHP into allopregnanolone by decreasing the Km value

of this enzyme for 5a-DHP by 10- to 30-fold. Even though

the molecular mechanisms of fluoxetine and norfluoxetine

action remain to be elucidated, the pharmacology of the

S stereoisomers of fluoxetine and norfluoxetine appears to

be prototypical for molecules that possess specific neuros-

teroidogenic activity [19–21]. Thus, these drugs, which

were originally termed ‘‘SSRI’’ antidepressants, may be

beneficial in psychiatric disorders because in doses that are

inactive on serotonin reuptake mechanisms, they increase

the bioavailability of neuroactive GABAergic steroids (for a

review see 21, 42, 45–50). Based on these considerations,

we have proposed that ‘‘SSRIs’’ should be changed to the

more appropriate term ‘‘selective brain steroidogenic stim-

ulants’’ (SBSSs), which more accurately defines the

pharmacological profile of fluoxetine and its congeners [21].

It is important to note that socially isolated adult mice

show a sexually dimorphic response. Female mice fail to

exhibit either 5a-RI expression downregulation or brain

allopregnanolone level downregulation [19, 51]. Hence,

female mice fail to express aggression or a decreased

§

00 1 2 3 4 6 8

50

Isolation Period (weeks)

****

0

5

10

15

§

Allopregnanolone

5α-reductase

Aggression 75

25

**

100

5α-R

I mR

NA

(at

tom

ol/µ

g R

NA

)

•

• • •

20§ §

Att

ack

Du

rati

on

(se

c/10

min

)

*

Allo

pre

gn

ano

lon

e (p

mo

l/g O

B)

Fig. 3 Time-course of aggressiveness development and allopreg-

nanolone content and 5a-reductase type I (5a-RI) mRNA expression

decrease during social isolation in male mice. Aggression of a

resident mouse against an intruder was measured as the duration of

attacks in 10 min. Allopregnanolone was determined in the olfactory

bulb (OB) and 5a-RI mRNA expression in the frontal cortex of the

same mice killed immediately after termination of the resident-

intruder test. Each value is the mean ± SEM of eight animals. *,

P \ 0.01 when 5a-RI mRNA expression at a given time period of

social isolation is compared with social isolation period 0. •,

P \ 0.01 when allopregnanolone content at a given time period of

social isolation is compared with social isolation period 0; §,

P \ 0.01 when the duration of attacks at a given social isolation

time period is compared with social isolation period 0 (Modified from

Pinna et al., [19])

Neurochem Res (2008) 33:1990–2007 1993

123

pentobarbital-elicited loss of righting reflex [19, 51, 52].

However, in socially isolated female mice, TP treatment

that downregulates the mRNA and protein expression of

5a-RI and results in a substantial decrease of brain allo-

pregnanolone levels elicits aggressive behavior and

modifies sexual behaviors [52]. Allopregnanolone and

S-norfluoxetine-elicited normalization of TP-induced brain

allopregnanolone level downregulation in female mice

stereoselectively abolishes abnormal behaviors [53].

Thus, the sexually dimorphic decrease of cortico-limbic

allopregnanolone content maintained by 5a-RI expression

downregulation may be an important contributor to deficits

in GABAA receptor neurotransmission in brain circuits that

mediate the behavioral effects of social isolation or TP

administration.

Behavioral Effects of AAS Abuse

Despite the dramatic increase in AAS abuse rates [54] and

the worldwide health concerns [55, 56], the high priority

given by NIDA to mechanistic investigations on AAS

abuse [57, 58] and a large body of evidence showing that

repeated treatment with AAS increases aggression in

humans as well as in laboratory animals [52–54, 56, 59],

the molecular mechanisms and neuronal circuitries

involved in AAS-induced behavioral symptoms still

require anatomical and biochemical investigation.

Growing evidence shows that AAS induce a wide range

of CNS effects. The positive effects include improved

mental acuity and increased sexual drive, but these are

associated with less wanted features such as impulsive

aggression, episodic mania, psychosis, and major depres-

sion [54, 60]. Episodes of irritability and impulsive

aggression may escalate to extreme violence, including

murder attempts [61, 62]. AAS withdrawal symptoms

include severe depression and suicide attempts [63].

Studies examining this issue showed that AAS display

compound-specific aggression potential. The most abused

AAS include TP, which activates aggression at low non-

anabolic doses, while nandrolone and stanozolol induce

aggression in mice only at high anabolic doses (Table 1).

In Table 1, we report a comparison of the effects of TP,

nandrolone, and stanozolol on gastrocnemius muscle

weight and aggressive behavior in mice. The gastrocne-

mius is a hormone-sensitive muscle that has long been

recognized as a myotropic marker of the anabolic activity

of steroids [64]. It is evident that at the non-anabolic dose

of 1.45 lmol/kg s.c., TP is highly efficacious in the

induction of aggression, while highly anabolic doses of

stanozolol fail to induce aggression (Table 1).

The disparity among AAS in their dose-related anabolic

versus behavioral effects bears further investigation. For

example, a better understanding of the molecular mecha-

nisms whereby AAS affect GABAergic synaptic function

in local cortico-limbic circuits that trigger aggression and

other behavioral deficits is required.

The Relationship Between GABAA Receptor Function

and Neurosteroid Biosynthesis in AAS-induced

Behavioral Deficits

Although several neurotransmitter systems have been

implicated in AAS-induced aggression (i.e., serotonin,

dopamine, neuropeptides) [65, 66], recent data suggest that

GABAA receptor-mediated neurotransmission dysfunction

may play a major role in eliciting the aggression induced by

protracted treatment with AAS [52, 53, 66–68]. AAS elicit

both acute and chronic changes in the GABAergic signaling

system. The immediate effects, such as decreases in anxiety

and an enhanced sense of well-being, arise from a direct

enhancement of forebrain GABAergic circuits. In contrast,

enhanced aggression, anxiety, and fear require chronic

exposure to AAS, which may lead to a downregulation of

GABAergic neurotransmission, including genomic mecha-

nism targeting [66]. This may also include a downregulation

of neurosteroid biosynthesis [49, 50] and/or changes in the

expression of GABAA receptor subunits [7, 53, 69].

Aggressive Behavior in Socially Isolated

and Testosterone Propionate (TP)-Treated Mice

Our studies have suggested that TP-treated mice exhibit

aggressive behavioral components reminiscent of if not

identical to those exhibited by socially isolated male mice

Table 1 Testosterone propionate (TP), nandrolone (ND), and stan-

ozolol (ST) effects on mouse aggressive behavior and muscle weight

Mice Aggression (sec/10 min) Gastrocnemius (mg)

Vehicle 10 ± 3.2 174 ± 8.3

TP 0.15 24 ± 4.5 180 ± 8.7

TP 1.45 35 ± 3.5* 189 ± 8.4

TP 14.5 63 ± 9.1** 194 ± 9.2

TP 43.5 68 ± 11** 239 ± 5.8**

ND 2.20 16 ± 3.6 190 ± 8.4

ND 22.2 28 ± 3.1* 204 ± 6.6*

ND 66.6 62 ± 10** 237 ± 7.5**

ST 0.21 5 ± 1.8 195 ± 7.8

ST 2.10 3 ± 1.4 216 ± 6.0**

ST 21.0 15 ± 8.4 243 ± 5.7**

ST 63.3 61 ± 9.7** 246 ± 6.9**

AAS were administered (lmol/kg s.c.) daily for 3 weeks. Mean ±

SEM 6 mice. * P \ 0.05; ** P \ 0.01 with vehicle

1994 Neurochem Res (2008) 33:1990–2007

123

(Table 2). Hence, using an animal model in which aggres-

sion is induced in male mice by 1–4 weeks of social

isolation stress, we have observed an increase of brain

testosterone during the first 3–7 days of social isolation

[70]. This finding suggests that the upregulation of brain

testosterone levels in the first week of social isolation may

be an important factor in the development of neurobio-

logical synaptic patterns involved in the social isolation

syndrome.

Similarly, female mice, which express brain testosterone

levels that are *1/3 of the levels measured in males

(Table 2), fail to become aggressive following a protracted

period of social isolation. However, female mice injected

with TP fail to display sexually dimorphic responses to

social isolation and they also show not only a dose- and

time-related aggressiveness toward a male mouse intruder

but also courtship behaviors to a naıve female mouse [52,

53]. These altered behaviors are heightened following an

ovariectomy in addition to TP treatment. Ovariectomy

alone failed to induce increased levels of aggression when

female mice were socially isolated for a period of 3 weeks

[52]. Protracted treatment with TP for 3 weeks brings

female brain testosterone levels to values similar to those of

male mice (Table 2 and [52]). Likewise, orchiectomized

mice express lower brain (*10%) testosterone levels and

fail to exhibit aggression during a period of protracted

social isolation; when administered TP (Table 2) in doses

that normalize brain testosterone levels, aggression is

reinstated (Table 2 and [52]).

In group housed male mice, TP treatment (1.45–

43.5 lmol/kg, once daily) for 14 days results in a time-

and dose-dependent development of territorial aggression

to a same-sex intruder. In Fig. 4, we report the expression

of aggressive behavior after 14 days of treatment with

TP, when aggressive behavior reaches its near maximal

intensity. To measure aggression levels, TP-treated mice

were placed in a cage for 24 h previous to exposure to an

untreated intruder male mouse. A few seconds after the

introduction of an intruder, the resident mouse typically

initiates aggressive contacts resembling those of socially

isolated mice. The aggressive behavior of resident group

housed TP-treated mice is characterized by an initial

pattern of exploratory activity around the intruder, fol-

lowed by rearing and tail rattle, and accompanied in a

few seconds by wrestling and/or violent biting attacks.

Figure 4 shows that TP doses of 43.5 lmol/kg s.c. induce

an increase in aggression that is only slightly higher

than the increase in aggression elicited by a dose of

14.5 lmol/kg s.c. Importantly, mice that have received

Table 2 TP effects on the relationship among duration of attacks against an intruder, 5a-RI mRNA and protein expression, progesterone (P),

allopregnanolone (Allo), and testosterone (T) levels in the brain of socially isolated (SI) and group housed (GH) male and female mice

Mice Aggression (sec/10 min) 5a-RI attomol/lg RNA 5a-RI/b-actin (OD ratio) P (pmol/g) Allo (pmol/g) T (pmol/g)

Male

GH + VH None 365 ± 21 12 ± 2.8 19 ± 5.9 16 ± 3.6 31 ± 4.8

SI + VH 68 ± 11 177 ± 15a 4.8 ± 1.2a 18 ± 6.4 7.1 ± 1.2a 35 ± 4.5a

SI + ORX 9.4 ± 7.2b n.d. n.d. n.d. 20 ± 2.8 3.4 ± 1.5a

SI + ORX + TP 61 ± 13 n.d. n.d. n.d. 5.2 ± 0.9a 46 ± 9.3

Female

GH + VH None 415 ± 41 n.d. 65 ± 15 14 ± 2.3 12 ± 4.1

SI + VH 1.2 ± 0.5 437 ± 35 16 ± 2.5 58 ± 13 16 ± 3.1 10 ± 5.0

SI + TP 75 ± 8.3c 268 ± 18 8.2 ± 1.3c 69 ± 20 8.2 ± 1.6c 39 ± 5.2c

Allopregnanolone, progesterone, and testosterone were determined in olfactory bulbs and 5a-reductase type I (5a-RI) mRNA and protein

expression in frontal cortex of SI and GH mice killed immediately after behavioral tests. Testosterone propionate (TP, 1.45 lmol/kg) was

administered daily for 3 weeks. a P \ 0.01 vs. GH male mice. b P \ 0.01 vs. SI male mice. c P \ 0.05, vs. SI female mice. d P \ 0.01 vs.

respective control group. (One-way ANOVA followed by Dunnett’s test). Mean ± SEM of 6 mice; n.d. = not determined; ORX = orchiec-

tomized; VH = vehicle. Modified from Pinna et al., [52]

TP dose treatment (µmol/kg)

Att

ack

Du

rati

on

(sec

/10

min

)

60

45

30

15

00 1.45 3.75 14.5 43.5

Fig. 4 Dose-response of aggression development in testosterone

propionate (TP)-treated male mice. TP (1.45–43.5 lmol/kg s.c.,

dissolved in oil) or vehicle were administered once daily for 14 days.

Aggression of a resident mouse against a same-sex intruder was

measured as the duration of attacks in 10 min test. Each value is the

mean ± SEM of 5 mice

Neurochem Res (2008) 33:1990–2007 1995

123

saline treatment fail to exhibit aggression to an intruder

(Fig. 4).

Changes in locomotor activity measures (horizontal and

vertical activity) have been reported to be an important

factor in altered aggression levels [71]. Our study showed

that TP treatment failed to alter motor activity [52].

Collectively, the data suggest that high brain testoster-

one levels are essential to facilitate the development of

aggressive behavior in response to a stressful condition,

such as the presence of an intruder in the home cage.

Altered Contextual Fear Responses in Socially Isolated

and TP-Treated Mice

A Pavlovian fear conditioned paradigm [22] was used to

investigate the disturbed emotional behaviors of socially

isolated and TP-treated mice.

Mice were exposed to a novel environment (i.e., context,

a training chamber) and were allowed to explore it for

2 min. After this time, they received an acoustic tone (i.e.,

conditioned stimulus, CS) (30 s, 85 DB) co-terminated with

an unconditioned stimulus (US) (electric footshock, 2 s,

0.5 mA). The tone plus the foot shock were repeated 3 times

every 2 min. After the last tone + shock delivery, mice

were allowed to explore the context for an additional minute

prior to removal from the training chamber (total of 8 min).

On re-exposure to the context 24 h later, mice displayed a

conditioned fear response, including sustained freezing

behaviors. Freezing behavior, defined by the absence of any

movement except for those related to respiration while the

animal was in a stereotyped crouching posture [22, 72], was

measured for 5 min without tone or footshock presentation.

The effect of social isolation stress and TP treatment on

contextual freezing behavior is similar (Table 3). In

socially isolated male mice, there was a time-related

increase of contextual freezing duration [22]. The increase

of contextual freezing time remains at a plateau (*70%)

after 6–8 weeks of social isolation [22]. A substantial

reduction of freezing time was recorded in mice repeatedly

exposed to the fear conditioning setup, with the socially

isolated mice showing a slower extinction time [22].

Socially isolated female mice that have received a long-

term TP treatment (1.45 lmol/kg s.c.) express a 50%

increase in conditioned fear responses (Table 3). Impor-

tantly, no difference in freezing behavior was recorded

during the training session among TP- and vehicle-treated

mice, suggesting that perception of the unconditioned

stimulus is not altered in these mice [22].

Contextual fear expression is dependent on hippocampal

function [73, 74] but the amygdala [75–78] and cortical

regions [79, 80] seem to be involved in the mediation of

fear responses. Failure to extinguish the conditioned fear

responses to the context is generally regarded as an index

of altered cortico-limbic neurotransmission that occurs in

altered emotional disorders [75, 81, 82], including post-

traumatic stress disorders [39] and in mouse models of this

disorder [22, 83].

Therefore, understanding the molecular mechanisms

underlying conditioned fear expression and extinction may

help to develop therapeutic strategies for emotional dys-

functions and posttraumatic stress disorders.

A TP Treatment Downregulates Brain 5a-RI mRNA

Expression

In socially isolated mice, aggression is associated with a

time-dependent downregulation of allopregnanolone bio-

synthesis (Table 2 and Fig. 3).

Based on previously published behavioral studies [53],

we selected a TP dose of 1.45 lmol/kg given s.c. (dis-

solved in oil) once daily for 3 weeks and then measured

5a-RI mRNA expression in several cortico-limbic struc-

tures. Figure 5 shows decreased mRNA expression for

5a-RI following TP treatment in female mice measured in

punches obtained from tissue slices of the olfactory bulb,

somato-sensory frontal cortex, amygdala, and hippocam-

pus. The striatum and cerebellum fail to show changes of

5a-RI mRNA expression following TP treatment. It is

noteworthy that the decrease of 5a-RI mRNA expression is

greater in the amygadala (*70%) than in the other brain

areas so far investigated.

Importantly, the brain structures that are involved in the

TP-induced biochemical alterations in female mice appear

to be the same ones affected in males during social isola-

tion [41]. Neurons of these brain structures are primarily

comprised of pyramidal glutamatergic neurons, which

Table 3 Altered fear conditioned responses in socially isolated and

TP-treated mice are reversed by administering S-norfluoxetine

(SNFLX)

Groups Freezing time

(sec/5 min) during

contextual test

Female GH mice + Vehicle 70 ± 7.8

Female SI mice + Vehicle 78 ± 8.2

Female SI mice + TP 120 ± 5.1*

Female SI mice + TP + SNFLX 91 ± 8.9

Male GH mice + Vehicle 68 ± 9.9

Male SI mice + Vehicle 121 ± 5.6**

Male SI mice + SNFLX 80 ± 4.1

Mean ± SEM of freezing time duration measured in 5 mice.

* P \ 0.05; ** P \ 0.01 when TP-treated socially isolated (SI)

female or SI male mice are compared with vehicle-treated female or

group housed (GH) male mice. SNFLX was given 45 min before

training test at the dose of 1.8 lmol/kg, i.p

1996 Neurochem Res (2008) 33:1990–2007

123

express high levels of steroidogenic enzymes [18]. We

hypothesized that the expression of 5a-RI in glutamatergic

neurons is specifically downregulated during TP treatment

and accompanies an impairment of GABAergic function

maintained by brain allopregnanolone downregulation.

Decreased GABAergic function mediated by a TP-

induced allopregnanolone biosynthesis downregulation in

brain areas of the cortico-limbic circuit may be an important

factor in the expression and maintenance of aggressive

behavior, altered fear responses, and changes in sexual

behaviors [52, 53, 70].

The Expression of 5a-RI mRNA is Specifically

Downregulated in Glutamatergic Cortico-limbic

Neurons that Regulate Aggression and Emotional

Instability

To evaluate whether 5a-RI expression is specifically

downregulated in glutamatergic cortico-limbic neurons of

mice receiving TP treatment, we used in situ hybridization

technology to measure the neuronal expression of 5a-RI

and 3a-HSD in various brain regions. We have also com-

bined in situ antisense and immunohistochemistry labeling

with specific antibodies (i.e., GAD65/67, VGLUT) and

confocal fluorescence microscopy to verify distribution of

the neurons expressing 5a-RI and 3a-HSD in these brain

structures.

To identify the type of neurons that express 5a-RI, after

the in situ hybridization procedure was terminated the

following antibodies were used for specific protein identi-

fication: (i) rabbit anti-GAD67/65, (ii) rabbit anti-

VGLUT2, (iii) rabbit anti-GFAP, (iv) rabbit anti-S-100b,

and (v) rabbit anti-rat 5a-RI. Following double in situ

hybridization and immunohistochemistry procedures, the

slices were incubated with Cy5-labeled goat anti-rabbit

IgG or Cy5-labeled goat anti-guinea pig IgG to produce red

fluorescent staining or Cy2-labeled streptavidin to produce

green fluorescent staining. The number of cells in which

green and red fluorescence colocalize compared with the

number of cells that express only green or only red fluo-

rescence was quantified with confocal microscopy.

Details of the experimental procedures and the speci-

ficity of the antisense probes and antibodies are reported in

Agis-Balboa et al. [18, 41].

In situ hybridization hystochemistry studies have

revealed that 5a-RI and 3a-HSD mRNA colocalize in the

cortex, hippocampus, and amygdala with vesicular gluta-

mate transporter 2 (VGLUT2) proteins, (Fig. 6A1–A3). In

the cortex, both 5a-RI and 3a-HSD mRNA are expressed in

the cell bodies and dendrites of layers II, III, V, and VI

glutamatergic pyramidal neurons (Fig. 6A4), [18, 41].

After TP treatment, the intensity of the 5a-RI mRNA in

situ hybridization signal is decreased (*25%) specifically

in cortical layer II/III pyramidal neurons (Fig. 7). How-

ever, this signal fails to change in layer V/VI pyramidal

neurons (Fig. 7). Neither the cortical pyramidal neurons of

layer II/III nor those of layer V/VI show the changes in

3a-HSD mRNA detected with in situ hybridization after

long-term TP treatment (not shown).

The intensity of the 5a-RI mRNA in situ hybridization

signals is decreased by *35% in CA3 glutamatergic

pyramidal neurons and in glutamatergic DG granule cells

(Fig. 7). However, the intensity of 5a-RI mRNA staining

does not appear to be changed in CA1 glutamatergic

pyramidal neurons (Fig. 7). The greatest decrease (*70%)

in 5a-RI mRNA expression occurs in glutamatergic neurons

of the basolateral amygdala but not in the central amygdala

(not shown). Interestingly, we failed to detect a decrease of

5a-RI mRNA expression in GABAergic output neurons

(Fig. 7) in the reticular thalamic nucleus or in the striatum.

The intensity of the 3a-HSD mRNA in situ hybridiza-

tion signal appears to be unaffected following TP treatment

in the hippocampus or amygdala (not shown).

Functional Significance of the Downregulation of 5a-RI

in Cortico-limbic Glutamatergic Circuits of TP-Treated

Mice.

Lateral and basal amygdaloid nuclei, which also receive

major excitatory projections from the CA1 and subiculum in

addition to cortical excitatory afferents [84], project to

central amygdaloid nucleus neurons via excitatory gluta-

matergic pyramidal-like neurons directed to the intercalated

GABAergic neurons [85]. Central amygdaloid nucleus

neurons, which are GABAergic, intensify this information

-30

Amy

30

-60

••

0FC

Str

Crb

•

OB

•

-90

% o

f ch

ang

es o

f 5α

-RI

mR

NA

exp

ress

ion

Hip

Fig. 5 Protracted TP treatment (1.45 lmol/kg/3 weeks, s.c.,) is

associated in female mice with brain region-specific 5a-reductase

type I (5a-RI) mRNA expression downregulation. 5a-RI mRNA

expression was measured in samples obtained by punchings from

several brain areas. Olfactory bulb (OB), somatosensory frontal

cortex (FC), amygdala (Amy), hippocampus (Hipp), striatum (Str),

and cerebellum (Crb). •, P \ 0.05; ••, P \ 0.01 with vehicle.

Mean ± SEM of 5 mice (one-way ANOVA followed by Dunnett’s

test). Data are expressed as percent of change of fmol 5a-RI/nmol

NSE mRNA

Neurochem Res (2008) 33:1990–2007 1997

123

and control the expression of behavioral, autonomic, and

hormonal (emotional) responses by way of projections to the

brainstem and hypothalamus [86, 87].

It has been proposed that cortico-hippocampal-amyg-

dala circuits normally allow organisms to adjust emotional

behaviors when environmental circumstances change [88,

89]. Furthermore, it appears that some alterations in these

circuits may contribute to the cortical or basolateral

amygdala loss of control over the central amygdala [12].

The inability of some subjects to regulate their emotions

may be intensified by the circuit dysfunction that contrib-

utes to the genesis of aggressive behavior, anxiety, and

impulsivity in humans [90]. Therefore, a reduction in 5a-RI

and allopregnanolone expression in the glutamatergic

neurons of the somatosensory frontal cortex, CA3, and

dentate gyrus in the hippocampus and basolateral amygdala

could impair the function of cortico-hippocampal-amyg-

daloid circuits and explain the aggressive behavior, fear,

and anxiety observed in socially isolated and TP-treated

mice [19, 21, 22, 41, 52, 53, 68].

Figure 7 shows that the CA3 and dentate gyrus regions in

the hippocampal formation and the basolateral amygdala

exhibit a large decrease of 5a-RI mRNA expression in

glutamatergic output neurons. One may infer that the crucial

neuronal substrates targeted by TP treatment that can induce

aggression and contextual fear memories are expressed in

these circuits. Clearly, the hippocampus and amygdala inter-

act with other neuronal structures of the limbic cortex and

olfactory bulb that have also been implicated in the brain

circuits involved in the mediation of emotional behaviors [91].

Hence in socially isolated and in TP-treated mice, the

reduction of 5a-RI mRNA expression and allopregnano-

lone content in selected glutamatergic neuronal population

of the cortico-limbic and cortico-thalamic circuits likely

contribute to anxiety and to emotional disorders.

The Brain Allopregnanolone Increase Induced by

Agents that Stimulate Neurosteroidogenesis Abolishes

Aggression and Exaggerated Fear Responses

Experiments studying the mechanisms operative in the

aggression of socially isolated and TP-treated mice have

shown that allopregnanolone dose-dependently attenuates

the duration of attacks toward a male intruder (21 and

Fig. 8a). The attenuation of aggression is accompanied by

an upregulation of cortico-limbic allopregnanolone content

(Fig. 8b). A single dose of S-norfluoxetine (1.8 lmol/kg

i.p.) normalizes brain allopregnanolone levels and reduces

aggression in mice that have been socially isolated or

received long-term treatment with TP (19 and Fig. 9).

40 microns

5α-RI mRNA VGLUT2 Protein Merged

20 microns

A4

3A 2A 1A

Fig. 6 5a-RI mRNA is

expressed in glutamatergic

pyramidal neurons of the frontal

cortex. A1, confocal image of

5a-RI mRNA in frontal cortex

layer V pyramidal neurons color

coded in green. A2, VGLUT2

protein in the same neurons,

color coded in red. A3, merge of

A1 and A2. In A4, note that 5a-

RI mRNA is also expressed in

the apical dendrites. Coronal

sections correspond roughly to

bregma +1.4 mm [137]

-30

30

-60 ••

0

•

-90

Layers II-III V-VI CA3 BLA StrCA1 DG

RtN

•% o

f ch

ang

es o

f 5α

-RI

mR

NA

exp

ress

ion

Fig. 7 5a-RI mRNA in situ hybridization signal in neurons of several

cortico-limbic structures obtained from TP (1.45 lmol/kg/3 weeks,

s.c., dissolved in oil)-treated mice. Student’s t test, •, P \ 0.05; ••,

P \ 0.01. Layers II–III and V–VI = cortical pyramidal neurons

layers II–III and V–VI; CA1 = hippocampal CA1; CA3 = hippo-

campal CA3; DG = dentate gyrus granular cells; BLA = basolateral

amygdaloid nuclei; Rt = reticular thalamic nucleus. Each value in the

percent of change of the mean ± O.D. SEM of 5 mice

1998 Neurochem Res (2008) 33:1990–2007

123

Either allopregnanolone (not shown) or S-norfluoxetine

treatment normalized the increased fear responses in

socially isolated or TP-treated mice (Table 3) and [22] but

failed to do so in group housed mice [22]. The dose of S-

norfluoxetine administered is about 10 times lower than the

dose required to inhibit 5-HT reuptake [20, 21], suggesting

that aggression is not regulated by modifications of 5-HT

uptake efficacy but instead by the upregulation of brain

allopregnanolone levels.

Inhibition of 5a-RI Activity with SKF 105,111

Decreases Allopregnanolone Levels in the Mouse Brain

and Induces Aggression and Altered Fear Responses

To provide further support to the hypothesis that cortico-

limbic allopregnanolone levels are important in the

regulation of aggressive behavior and conditioned fear

responses in socially isolated male mice and in female

mice treated with TP, we gave the potent 5a-RI inhibitor

SKF 105,111 to group housed mice.

SKF 105,111 injections are highly efficacious in rapidly

(*1 h) decreasing the levels of allopregnanolone in the

olfactory bulb, frontal cortex, hippocampus, and amygdala

[22, 25]. This endogenous brain allopregnanolone decrease

downregulates GABAA receptor responsiveness to the

positive allosteric modulators of GABA action at GABAA

receptors, including pentobarbital, muscimol, ethanol, and

benzodiazepine, or to GABAA receptor antagonists such as

picrotoxin [25, 29, 38, 92]. A potential direct modulatory

effect of SKF 105,111 on GABAA receptors was ruled out

[25, 26].

Allopregnanolone content was measured in the mouse

frontal cortex prepared 15, 30 min, and 1, 3, 6, and 24 h

after i.p. administration of 48 lmol/kg of SKF 105,111.

Figure 10 shows that frontocortical allopregnanolone con-

tent was already decreased by *40% after 30 min and by

*80% at 1 h after SKF 105,111 treatment and reached a

maximum decrease of approximately 90% between 2 h and

6 h post SKF 105,111 injection. Thus, this SKF 105,111-

mediated brain allopregnanolone content decrease was

maintained for at least 6 h. Figure 11 shows that aggressive

behavior in mice is induced following a single injection of

SKF 105,111 (48 lmol/kg s.c.). Aggressive behavior was

assessed 2 h following each SKF injection in a 3-day SKF

treatment.

Consistent with the abovementioned results, treatment

with SKF 105,111 administered 2 h before training

induced a dose-dependent increase of fear conditioning

responses that negatively correlated with hippocampal

allopregnanolone level depletion [22].

•

Allopregnanolone

Aggression

15

0

10

Allo

pre

gn

ano

lon

e

(Pm

ol/g

)

Du

rati

on

of

Att

acks

Ag

ain

st

Intr

ud

er (

sec/

10 m

in)

Allopregnanolone (µmol/kg, i.p.) (µmol/kg, i.p.)

75

50

25

00 4 8 0 8 016

•

20

5

VHtreated

†

Testosterone propionate treated

BA

OB Allo content

Fig. 8 Allopregnanolone dose-dependently decreases the duration of

aggression in testosterone propionate (TP)-treated (1.45 lmol/kg/

3 weeks, s.c., dissolved in oil) female mice (a). Allopregnanolone

(Allo) in doses of 8 lmol/kg upregulates olfactory bulb (OB)

allopregnanolone levels (b). Mean ± SEM, n = 5, •, �, P \ 0.05

with TP + VH (0) (one-way ANOVA followed by Dunnett’s test).

Modified from Pibiri et al. [53]

Du

rati

on

of

Att

acks

(se

c/10

min

)

30

60

TP+SNFLX

Allo

pre

gn

ano

lon

e (P

mo

l/g O

B)

0

5

10

15

20

Vehicle

•

TP

0

90

•

••

Fig. 9 S-norfluoxetine (SNFLX) reduces aggression by upregulating

olfactory bulb (OB) allopregnanolone content in protracted TP-treated

(1.45 lmol/kg/3 weeks, s.c.) mice. Mean ± SEM of 5 mice.

S-norfluoxetine (S-NFLX, 1.8 lmol/kg, i.p.) was given 30 min before

the resident-intruder test. •, P \ 0.01 vehicle and S-NFLX-treated

with TP groups (one-way ANOVA followed by Dunnett’s test)

5.0

•

Allo

pre

gn

ano

lon

e

(Pm

ol/g

Fro

nta

l Co

rtex

)

6.0

20 151051 20.50

Time after SKF 105,111 administration (hr)

0

4.0

3.0

2.0

1.0

•••

25

Fig. 10 Time-dependent decrease in cortical allopregnanolone fol-

lowing a single intraperitoneal injection of SKF 105,111 (48 lmol/

kg). Mice were killed 0.25, 0.5, 1, 3, 6, and 24 h after SKF 105,111

administration. Mean ± SEM of 5 mice. •, P \ 0.01 compared to the

value at t = 0 (one-way ANOVA followed by Dunnett’s test)

Neurochem Res (2008) 33:1990–2007 1999

123

The use of SKF 105,111 in reducing allopregnanolone

and increasing aggression and contextual fear suggests that

effects of excess testosterone on aggression and fear

responses are in fact due to effects on allopregnanolone

rather than a direct effect of testosterone or of metabolites.

Possible Mechanisms Involved in AAS-induced

Allopregnanolone Biosynthesis Downregulation

Ongoing studies in our laboratory have indicated a disparity

in the potency of TP or other AAS, such as stanozolol and

nandrolone, in inducing anabolic versus behavioral effects

(Table 1). For example, stanozolol, which produces a

stronger selectivity for anabolic effects, induces aggressive

behavior only at the high dose of 66 lmol/kg/day for

3 weeks (Table 1). Of note, stanozolol and nandrolone are

not substrates for aromatase; whereas nandrolone is weakly

reduced, stanozolol is an alkylated steroid that cannot be

reduced by 5a-RI [93, 94]. Importantly, nandrolone and

stanozolol along with TP share the common ability to bind at

androgen receptors [93], raising the hypothesis that these

AAS may transcriptionally regulate 5a-RI through a specific

androgen receptor-mediated mechanism. This mechanism

does not appear to require either aromatization or reduction

of AAS as suggested by the results presented in this study

and published reports: (a) following long-term (1–3 week)

TP treatment, aggressive mice fail to express increased

levels of brain 5a-DHT or estradiol; and (b) stanozolol and

nandrolone increase aggression (Table 1) but fail to be

aromatized or to be consistently reduced [93, 94]. It is

remarkable that the rank of affinity for androgen receptors

for these AAS (testosterone [ nandrolone [ stanozolol)

[93] parallels the aggression liability of these substances

(testosterone [ nandrolone [ stanozolol) (Table 1).

Proof for the hypothesis that AAS may transcriptionally

regulate cortico-limbic 5a-RI expression through a specific

androgen receptor-mediated mechanism can be obtained by

administering androgen receptor antagonists, such as flu-

tamide or bicalutamide, to AAS-treated mice. It is expected

that these drugs may block AAS-induced aggression as

well as the molecular events that ultimately give rise to a

cortico-limbic allopregnanolone downregulation.

In addition to the action of TP on brain 5a-RI expres-

sion, there is also an alternative mechanism to be

considered—the competition of TP with progesterone for

5a-RI catalytic activity [95]. Establishing the influence of

precursor utilization and the competition for steroidogenic

enzymes in the periphery (i.e., plasma) and in the brain by

measuring the levels of allopregnanolone, 5a-DHP, and

progesterone and also testosterone, 5a-DHT, and estradiol

may suggest whether the levels of testosterone and/or

5a-DHT reach concentrations able to compete for 5a-RI

and/or 3a-HSD. By this mechanism, TP could decrease

brain allopregnanolone levels in addition to constitutively

decreasing 5a-RI expression [52, 96]. 5a-RI is very abun-

dant in the brain and liver whereas 5a-RII is highly

expressed in the prostate but is very low in the brain

[96–99]. Based on results showing that 5a-DHT fails to

increase in the brain following TP treatment (not shown), it

is expected that prostate 5a-RII and liver 5a-RI will reduce

peripheral testosterone into 5a-DHT, which may not be

taken up from the brain as reported previously for neu-

rosteroids that are synthesized and act in the brain in a

manner unrelated to peripheral renovation sources [23, 24,

32]. However, the limitations of our studies were in the

measurement of testosterone and 5a-DHT 24 h post-

injection following a 3 weeks treatment with TP. Studying

testosterone, progesterone, and respective metabolite levels

at different times following a single TP injection will give

more accurate information on the issue of substrate com-

petition for 5a-RI. The ratio between the levels of

testosterone and 5a-DHT or progesterone and 5a-DHP and

allopregnanolone in the brain and periphery will provide

important information relevant to the mechanism of allo-

pregnanolone level downregulation by testosterone in

selected cortico-limbic structures.

However, our results seem to disprove the hypothesis

that TP-induced aggression and brain allopregnanolone

level decreases are the result of a substrate competition

with progesterone in the utilization of 5a-RI. In addition to

the results described above for AAS, which induce

aggression but cannot be reduced and therefore fail to

compete with progesterone in utilizing 5a-RI, the following

considerations could be entertained: (1) a single dose of TP

fails to decrease allopregnanolone levels or to induce

10

40

20

30

00 1

Days of treatment

Vehicle

SKF 105,111

Att

ack

Du

rati

on

(se

c/10

min

) •••

3

Fig. 11 Time-course of aggression development in resident male

mice against an intruder. SKF 105,111 (48 lmol/kg s.c.) or vehicle

were given once daily 2 h prior testing. Each value is the mean ±

SEM of 5 mice. •, P \ 0.01 SKF 105,111-treated with vehicle group

(one-way ANOVA followed by Dunnett’s test)

2000 Neurochem Res (2008) 33:1990–2007

123

aggression (Fig. 3); however, (2) SKF 105,111, a potent

competitive inhibitor that rapidly blocks 5a-RI, thereby

depleting brain allopregnanolone content by approximately

70% in 1 h (Fig. 10) consistently induces a rapid expres-

sion of aggression (Fig. 11); also, (3) several weeks

following discontinuation of protracted TP treatment,

aggression is still high (data not shown), although at this

time drug levels are no longer present in the brain [100];

and (4) TP treatment results in testosterone concentrations

in the brain that are in the low nM range measured 24 h

post-injection (Table 2). These concentrations are at least

10-fold lower than the concentrations required to reach the

Km of 5a-RI for substrate, which is 1–5 lM [101].

Significance

AAS have become a major public health concern world-

wide because this recreational drug use occurs not only in

adolescents of both sexes but also in older subjects [54, 57,

58, 102]. Protracted AAS use in humans has been linked to

indiscriminate unprovoked aggression and violence [55].

AAS use intensifies the display of verbal aggression,

increases violence toward women, and increases the like-

lihood of homicidal actions [63]. However, studies of AAS

abuse in humans have several shortcomings. For example,

AAS abusers ‘‘stack’’ (that is, administer several AAS in

various ways) [54, 57, 58], or AAS may be taken with other

drugs of abuse, making an unambiguous assessment of

drug actions impossible. Studies using animal models are

advantageous because the inherent variables are controlled.

TP induces aggressive behavior in mice over protracted

administration at doses that approximate the TP dose range

used in humans (Fig. 4), and [52, 53, 56].

In TP-treated mice, aggression induction may involve a

decrease of the allopregnanolone content that is specifically

synthesized in glutamatergic neurons of cortico-limbic

structures. Our results suggest that protracted exposure to

TP may decrease the tone of GABAA receptors located in

glutamatergic neurons by reducing the bioavailability of

allopregnanolone in local brain circuits that regulate

aggression, fear, and sexual responses.

Submicromolar doses of the highly potent SBSS fluox-

etine and its congeners (e.g., S-norfluoxetine) increase

allopregnanolone content in selected areas of the mouse

brain (olfactory bulb, frontal cortex, amygdala, but not the

striatum and cerebellum) [19, 33] and attenuate TP-induced

aggression [52, 53]. This pharmacology relates to doses

that fail to inhibit 5-HT reuptake [19–21] but stereospe-

cifically upregulate brain allopregnanolone content.

Understanding the contribution of increase of local brain

neurosteroid biosynthesis to the specific brain circuitry of

neuronal populations that regulate aggression may have

important implications for the design of new non-sedative

treatments for the therapeutic control of AAS-induced

behavioral deficits. By improving the response of GABAA

receptors to GABA, drugs that selectively modify allo-

pregnanolone levels expressed locally in specific cortico-

limbic circuitry may help to establish new therapeutics for

more appropriate management of the impulsive aggression,

irritability, irrational fear, anxiety, and depression that

result from AAS abuse.

Future Studies

This review is focussed on: (a) the pivotal role of cortico-

limbic allopregnanolone levels in the regulation of social

isolation and AAS-induced aggressive behavior; (b) the

circuitry that express an allopregnanolone biosynthesis

downregulation during social isolation or TP treatment;

and (c) the mechanisms by which TP downregulates cor-

tico-limbic allopregnanolone levels, which can be the

result of a 5a-RI expression downregulation or of the

competition of testosterone with progesterone for the cat-

alytic activity of 5a-RI.

Several additional studies are required to better under-

stand the molecular mechanisms involved in the behavioral

dysfunctions caused by AAS:

Effects of Several Classes of AAS on Aggression

and Brain Allopregnanolone Levels

Because protracted TP treatment elicits a downregulation

of 5a-RI mRNA expression only in a neuron-specific

manner in selected brain structures, and these changes are

correlated with the development of aggression, it would be

important to establish whether the cellular expression of

5a-RI and perhaps also 3a-HSD is changed in the same

manner using different types of AAS. Comparison of

aggression expression and neurosteroid biosynthesis

downregulation elicited by TP, nandrolone, and stanozolol

in target circuits could reveal a common mechanism by

which long-term AAS abuse elicits behavioral alterations.

Because a likely mechanism of action for AAS to induce

aggression is the transcriptional regulation of 5a-RI

through a specific androgen receptor-mediated mechanism,

a thorough study of the effects of several AAS in a larger

number of brain areas that express androgen receptors

would be also required.

Impact of AAS on Peripheral Expression

of Allopregnanolone Biosynthesis

In future studies to understand the mechanisms by which

AAS induce behavioral abnormalities, it would also be

Neurochem Res (2008) 33:1990–2007 2001

123

important to investigate the effects of TP and possibly

other AAS on the peripheral expression of 5a-RI and

3a-HSD. Further, study of peripheral and brain levels of

steroids that appear to be directly involved in AAS-induced

aggression would be needed. Together with the levels

of the precursor of allopregnanolone and metabolites of

testosterone, these studies would provide important infor-

mation on the central and peripheral regulation of

steroidogenic enzymes by TP.

AAS Effects on Hormonal Systems

The effects of AAS on the neuroendocrine system are very

important in understanding the mechanisms by which

protracted AAS induce aggression. AAS have been repor-

ted to dramatically impact several hormonal systems, most

notably the hypothalamic-pituitary-adrenal (HPA), hypo-

thalamic-pituitary-thyroid (HPT), and hypothalamic-

pituitary-gonadal (HPG) axes [103–107]. These hormonal

axes are known to influence mood and behavior and their

perturbations in association with affective disorders and

other psychiatric conditions are deemed pathophysiologi-

cally relevant [107]. Thus, changes in neuroendocrine

function secondary to AAS intake appear to contribute to

AAS-induced psychiatric conditions [107].

High dosage AAS administration profoundly affects

hormonal balance in the HPG and HPT axes but has little

impact on the HPA axis. In the HPG axis, AAS decrease sex

hormone binding globuline (SHBG) and testosterone levels

and suppress gonadotropin levels [105, 107, 108]. Addi-

tionally, decreases in estradiol levels have been reported

[107]. In the HPA axis, some studies have reported

increased cortisol levels [104] and decreased levels of

dehydroepiandrosterone (DHEA) [109] and ACTH [104]

during AAS administration. In the HPT axis, AAS decreases

thyroid binding globulin, thyroxin (T4), and thriiodothyro-

nine (T3) [103, 105–108]. Altered thyroid function has long

been associated with acute psychiatric illness and changes in

thyroid hormone levels have been associated with the

increased aggressiveness induced by AAS [107].

Several reports have also established an important

relationship between thyroid hormones and androgen lev-

els. As discussed above, androgens may not only regulate

the production of thyroid hormones, but thyroid hormones

have also been shown to induce steroidogenic acute regu-

latory (StAR) protein expression, a key enzyme in the

neurosteroidogenic cascade, which results in neurosteroid

(i.e., allopregnanolone) production [110–113] (Fig. 2).

Remarkably, several similarities exist between thyroid

hormones and neurosteroids in the brain. First, thyroid

hormones, including T3 and metabolites (3,3’-diiodothyr-

onine and 3,5-diiodothyronine), similar to neurosteroids

[18, 25, 28, 32, 33], are produced in various brain areas

[114–118]. Further, similar to neurosteroids [32], the

metabolism of thyroxin (T4) in the CNS is subject to a

highly specific regulatory mechanism that differs substan-

tially from that described in peripheral tissues such as the

liver or kidney. In peripheral tissues, most of the active

iodothyronine T3 is taken up directly from the blood,

whereas the supply of T3 to the brain depends almost

completely on the cellular uptake and intracellular deio-

dination of T4 [119]. This implies that the supply of T4 and

intracellular deiodination are essential for the function of

T3 in the CNS. Also, like neurosteroids [21, 39, 40, 42, 45–

50], reports in the literature have suggested a role for T3 in

several psychiatric conditions, including depression and

affective disorders [120] and in animal models of these

disorders [121]. In humans, antidepressant actions are

highly facilitated when antidepressants are administered in

combination with T4 (reviewed in 120, 122). Additionally,

as observed in neurosteroids [19, 21, 33, 42], T3 levels in

rodent cortico-limbic structures are increased by several

antidepressant agents, mood stabilizers, and sleep depri-

vation (a fast acting antidepressant tool) [121].

It is therefore conceivable that the mechanism of action

of AAS-induced behavioral deficits may also include a

AAS-induced impairment of hormonal systems, including

the HPT axis, that are likely to impact cortico-limbic

allopregnanolone biosynthesis.

Regulation of 5a-RI and/or 3a-HSD through an

Epigenetic Promoter Hypermethylation Mechanism

In TP-treated mice, the decrease of 5a-RI mRNA expres-

sion and very likely the decrease of allopregnanolone

content in selected populations of glutamatergic neurons of

the somatosensory frontal cortex, hippocampus, and

amygdala allow inferences on the nature of synaptic cir-

cuits that are directly linked to these abnormal behavioral

responses. However, the present reports do not address the

molecular mechanisms responsible for 5a-RI expression

downregulation.

Recently, it has been shown that the 5’ upstream region

of the rat 5a-RI includes all the features of CpG islands and

contains several potential binding sites for the transcription

factor Sp1, which has been implicated in the activation of a

very large number of genes [123–125]. Sp1 is thought to be

involved in the epigenetic control of the promoter activity

regulating chromatin remodeling and favoring the propa-

gation of methylation-free islands on gene promoters [126].

From these data, it is possible to infer that in the mouse, the

expression of 5a-RI could also be regulated through an

epigenetic regulation of promoter hypermethylation that

may trigger 5a-RI mRNA downregulation after TP treat-

ment or following long-term social isolation. In favor of

this hypothesis are data from 3-week socially isolated

2002 Neurochem Res (2008) 33:1990–2007

123

aggressive mice showing that they express a significant

increase in the expression levels of fronto-cortical DNA

methyltransferase-1 (DNMT1) mRNA [127]. DNMT1 is

abundantly expressed in GABAergic neurons of the mouse

and human brain, where it catalyses the transfer of methyl

groups from S-adenosyl methionine (SAM) to cytosine

residues in promoters embedded in CpG islands [128, 129].

This finding is consistent with a social isolation-induced

hypermethylation of the 5a-RI promoter, consequent

downregulation of brain 5a-RI mRNA expression and

allopregnanolone levels, and increased aggression.

In this case, it is suggested that histone deacetylase

inhibitors such as valproate or MS-275 [130–132], inhibi-

tors of DNMT1 such as zebularine or procainamide [133,

134], or DNA-demethylase inducers such as valproate or

MS-275 [129, 134] should be considered as drugs that could

be tested to reverse the 5a-RI expression downregulation

induced by TP or social isolation. Characterization of these

drug activities should also be advantageous in the treatment

of depression and posttraumatic stress disorder because in

these psychiatric disorders, allopregnanolone brain levels

are downregulated [39, 40], probably because 5a-RI and/or

3a-HSD expression are epigenetically downregulated.

It would be of interest to evaluate the mechanisms

orchestrating this complex epigenetic response. Drugs that

affect epigenetic mechanisms, such as valproate, in com-

bination with antipsychotics are currently being tested in

clinical trials to treat psychiatric illness, such as schizo-

phrenia, bipolar disorder, depression, and drug addiction

[135, 136].

Although the molecular mechanisms that underlie the

action of these drug associations are not fully understood,

ultimately findings from TP-treated mice or socially iso-

lated mice receiving these drugs will generate insights into

new molecular targets for the treatment of psychiatric

disorders, such as anxiety and panic disorders, depression,

psychosis, and posttraumatic stress disorders.

Acknowledgment This study was supported by a Campus Research

Board Award 2-611185 (to GP). Supported by Regione Autonoma

della Sardegna, Italy, ‘‘Master and Back’’ (to F.P.).

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