Progress in Neurobiology 113 (2014) 79–87

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Progress in Neurobiology
journal homepage: www.elsevier.com/locate/pneurobio

The role of allopregnanolone in depression and anxiety

Cornelius Schüle a,*, Caroline Nothdurfter b, Rainer Rupprecht b
a
Department of Psychiatry and Psychotherapy, Ludwig-Maximilian-University, Nussbaumstr. 7, 80336 Munich, Germany
b
Department of Psychiatry and Psychotherapy, University Regensburg, Universitätsstrasse 84, 93053 Regensburg, Germany

A R T I C L E I N F O A B S T R A C T

Article history: Neuroactive steroids such as allopregnanolone do not only act as transcriptional factors in the regulation
Received 4 June 2013 of gene expression after intracellular back-oxidation into the 5-a pregnane steroids but may also alter
Received in revised form 21 September 2013 neuronal excitability through interactions with specific neurotransmitter receptors. In particular, certain
Accepted 21 September 2013
3a-reduced metabolites of progesterone such as 3a,5a-tetrahydroprogesterone (allopregnanolone) and
Available online 8 November 2013
3a,5b-tetrahydroprogesterone (pregnanolone) are potent positive allosteric modulators of the GABAA
receptor complex. During the last years, the downregulation of neurosteroid biosynthesis has been
Keywords:
intensively discussed to be a possible contributor to the development of anxiety and depressive disorder.
Allopregnanolone
Reduced levels of allopregnanolone in the peripheral blood or cerebrospinal fluid were found to be
Neuroactive steroids
Depression associated with major depression, anxiety disorders, premenstrual dysphoric disorder, negative
Anxiety symptoms in schizophrenia, or impulsive aggression. The importance of allopregnanolone for the
Affective disorders regulation of emotion and its therapeutical use in depression and anxiety may not only involve
GABAergic mechanisms, but probably also includes enhancement of neurogenesis, myelination,
neuroprotection, and regulatory effects on HPA axis function. Certain pharmacokinetic obstacles limit
the therapeutic use of natural neurosteroids (low bioavailability, oxidation to the ketone). Until now
synthetic neuroactive steroids could not be established in the treatment of anxiety disorders or
depression. However, the translocator protein (18 kDa) (TSPO) which is important for neuroster-
oidogenesis has been identified as a potential novel target. TSPO ligands such as XBD 173 increase
neurosteroidogenesis and have anxiolytic effects with a favorable side effect profile.
ß 2013 Published by Elsevier Ltd.

Contents

1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2. Allopregnanolone and depression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
2.1. Allopregnanolone in animal models of depression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
2.2. Allopregnanolone and clinical studies in depressed patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3. Allopregnanolone and anxiety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
3.1.1. Allopregnanolone in animal models of anxiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
3.2. Allopregnanolone and clinical studies in patients suffering from anxiety disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4. Conclusions and future directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

1. Background one receptor active 5 alpha-pregnane steroids (Rupprecht et al.,

Neuroactive steroids act as transcriptional factors in the
regulation of gene expression. The induction of DNA binding and
transcriptional activation of the progesterone receptor requires
intracellular oxidation of the neuroactive steroids into progester-
* Corresponding author. Tel.: +49 89 5160 5335.
E-mail address: Cornelius.Schuele@med.uni-muenchen.de (C. Schüle).
1993). However, neuroactive steroids may also alter neuronal
excitability through interactions with specific neurotransmitter
receptors (Rupprecht and Holsboer, 1999; Rupprecht, 2003).
These steroids are synthesized from cholesterol or steroidal
precursors (Fig. 1). The formation of pregnenolone from choles-
terol is regulated by the translocator protein TSPO (18 kD),
formerly called peripheral or mitochondrial benzodiazepine
receptor, which is mainly located in the outer mitochondrial
membrane and favors the transport of cholesterol to the inner

0301-0082/$ – see front matter ß 2013 Published by Elsevier Ltd.

http://dx.doi.org/10.1016/j.pneurobio.2013.09.003
80 C. Schüle et al. / Progress in Neurobiology 113 (2014) 79–87

Fig. 1. Synthesis of neurosteroids (Nothdurfter et al., 2012a). The cholesterol side-chain-cleaving cytochrome-P-450 enzyme (P-450scc, CYP11A1) is located at the inner
mitochondrial membrane and converts cholesterol to pregnenolone. In the cytoplasm (diffusion marked by black arrow), pregnenolone is metabolized to progesterone by the
microsomal 3b-dehydrogenase/D5–D4 isomerase. Progesterone is further converted to deoxycorticosterone by the 21-hydroxylase (CYP21B). The reduction of progesterone
and deoxycorticosterone by the 5a-reductase leads to 5a-dihydroprogesterone and 5a-dihydrocorticosterone (5a-DHDOC). The neurosteroids allopregnanolone and
allotetrahydrodeoxycorticosterone (3a, 5a-tetrahydrodeoxycorticosterone, 3a, 5a-THDOC) are formed by further reduction through 3a-hydroxysteroid dehydrogenase
(3a-HSD).

mitochondrial membrane, thereby promoting neurosteroido-
genesis (Papadopoulos et al., 2006; Rupprecht et al., 2010).
Pregnenolone is further converted into an array of different
steroids (Fig. 1). Progesterone may be formed by the 3b-
hydroxysteroid dehydrogenase and serves as the main precursor
molecule for 3a-reduced neuroactive steroids. Progesterone and
deoxycorticosterone (DOC) are irreversibly reduced by the 5a-
reductase into 5a-dihydroprogesterone (5a-DHP) and 5a-dihy-
drodeoxycorticosterone (5a-DHDOC). These pregnane steroids
may be further reduced to 3a,5a-tetrahydroprogesterone
(3a,5a-THP; 3a-hydroxy-5a-pregnan-20-one; allopregnano-
lone), 3a,5b-tetrahydroprogesterone (3a,5b-THP; 5b-pregnan-
3a-ol-20-one; pregnanolone) and 3a,5a-tetrahydrodeoxycorti-
costerone (3a,5a-THDOC; 3a,21-dihydroxy-5a-pregnan-20-
one; allotetrahydrodeoxycorticosterone) by the 3a-hydroxyster-
oid dehydrogenase (3a-HSD).
In particular, certain 3a-reduced metabolites of progesterone
such as 3a,5a-THP (allopregnanolone) and 3a,5b-THP (pregna-
nolone) are potent positive allosteric modulators of the GABAA
receptor complex, whereas 3b,5a-THP or DHEA exert an inhibitory
modulation of the GABAA receptor (Paul and Purdy, 1992;
Rupprecht and Holsboer, 1999). In preclinical studies at the
molecular level, selective serotonin reuptake inhibitors (SSRIs)
were able to enhance the formation of 3a-reduced neuroactive
steroids (e.g. allopregnanolone) via shifting the activity of the
cytosolic 3a-HSD type 3 toward the reductive direction; only
sertraline also inhibited the reverse oxidative reaction. The
tricyclic antidepressant imipramine had no effect on the formation
of allopregnanolone (Griffin and Mellon, 1999). The authors of this
study hypothesized that these results may explain the rapid
amelioration of dysphoria and anxiety symptoms associated with
major depression and late luteal phase dysphoria by SSRIs.
However, another study, which did not find any change in the
activity of this enzyme after administration of various SSRIs, came
to the conclusion that the interference with the 3a-HSD type 3
might not be the crucial mechanism by which SSRIs increase
allopregnanolone concentrations (Trauger et al., 2002).
In our research group we examined the effects of the
antidepressant drug mirtazapine (a2-, 5-HT2-, 5-HT3- and H1-
antagonist) on the reduction of 5a-DHP (into 3a,5a-THP = allo-
pregnanolone) catalyzed by the cytosolic 3a-HSD type 3 and on
the oxidation of 3a,5a-THP (into 5a-DHP) catalyzed by the
microsomal 3a-HSD (Schule et al., 2006b). Pure recombinant
human cytosolic 3a-HSD type 3 protein was expressed in
Escherichia coli, whereas the cDNA for human microsomal 3a-
HSD was stably expressed in HEK-293 cells. Our in vitro
investigations demonstrated a dose-dependent inhibitory effect
of mirtazapine on the activity of the microsomal 3a-HSD in the
oxidative direction (conversion of 3a,5a-THP into 5a-DHP);
however, in contrast to SSRIs no effect of mirtazapine on the
cytosolic 3a-HSD catalyzing the reductive pathway (conversion of
5a-DHP into 3a,5a-THP) was observed. Nevertheless, these in
vitro investigations support the view that both SSRIs and
mirtazapine may enhance the formation of 3a-reduced neuroac-
tive steroids such as allopregnanolone thereby potentiating the
GABAergic tone in the brain. The effects of psychotropic drugs on
neurosteroidogenesis in humans, in particular depressed patients,
are summarized in Table 1.
 C. Schüle et al. / Progress in Neurobiology 113 (2014) 79–87 81

Table 1
Effects of psychotropic drugs on neurosteroidogenesis in humans (in particular depressed patients) (for details see Schule et al., 2011).

Drugs Possible mechanism of action Effects of neurosteroidogenesis

SSRIs Shifting of the activity of 3a-HSD toward No change in 5a-DHP; increase of 3a-THP (allopregnanolone),
the reductive direction but decrease of of 3a-THDOC; decrease of 3b-THP; in some studies
decrease of DHEA-S
TCAs Unclear (no effect on 3a-HSD) No change in 5a-DHP; increase of 3a-THP (allopregnanolone), no effect
on 3a-THDOC; decrease of 3b-THP; in some studies decrease of DHEA-S
Mirtazapine Inhibition of the oxidative pathway catalyzed Weak but significant increase in 5a-DHP; increase of 3a-THP
by the microsomal 3a-HSD (allopregnanolone), no effect on 3a-THDOC; decrease of 3b-THP;
reduction of both cortisol and DHEA-S (no impact on cortisol/DHEA-S-ratio)
Lithium Unclear Given as an additional treatment reversal of increase in 5a-reduced
neuroactive steroids as compared to mirtazapine monotherapy
Carbama-mazepine Unclear (may be enzyme induction via Given as an additional treatment reversal of increase in 5a-reduced
Cytochrom P450 system) neuroactive steroids as compared to mirtazapine monotherapy
SD – No effect on neurosteroidogenesis
rTMS – No effect on neurosteroidogenesis
ECT – No effect on neurosteroidogenesis
TSPO-ligands (e.g. XBD 173) TSPO facilitates the transport of cholesterol o General stimulation of neurosteroidogenesis
the inner mitochondrial membrane

Abbreviations: SSRIs, selective serotonin reuptake inhibitors; TCAs, tricyclic antidepressants; 3a-HSD, 3a-hydroxysteroid-dehydrogenase; 5a-DHP, 5a-dihydroprogester-
one; 5a-THP, 5a-tetrahydroprogesterone; 3a-THDOC, 3a-tetrahydroprodeoxycorticosterone; DHEA, dehydroandrostendione; SD, sleep deprivation; rTMS, repeated
transcranial magnetic stimulation; ECT, electroconvulsive therapy; TSPO, translocator protein (18 kDa).

During the last 15 years, the downregulation of neurosteroid
biosynthesis has been implicated as a possible contributor to the
development of anxiety and depressive disorder (Nothdurfter
et al., 2012a; Rupprecht et al., 2010; Schule et al., 2011). It could be
shown in quite a number of clinical trials that reduction in the
serum/plasma and cerebrospinal fluid content of neuroactive
steroids, in particular the GABAA receptor-agonistic progesterone
metabolite allopregnanolone, may be associated with several
affective disorders, including major depression (Romeo et al.,
1998; Schule et al., 2006b; Uzunova et al., 1998), anxiety disorders
(Rupprecht et al., 2009; Strohle et al., 2002), premenstrual
dysphoric disorder (Amin et al., 2006; Backstrom et al., 2003;
Pearlstein, 2010; Rapkin et al., 1997), postpartum depression
(Bloch et al., 2000; Nappi et al., 2001), posttraumatic stress
disorder (Rasmusson et al., 2006), negative symptoms in schizo-
phrenia (Marx et al., 2009, 2011), or impulsive aggression (Nelson
and Pinna, 2011). These findings gave reason to the hypothesis that
downregulation of allopregnanolone and other GABAergic neuro-
active steroids in the brain could be a risk factor for the
development of the mentioned psychiatric disorders, but may
be also involved in the pathophysiology of psychiatric and
neurological disorders such as epilepsy (Herzog, 1995; Reddy,
2011), learning and memory deficit disorders such as Alzheimer’
disease (Luchetti et al., 2011), or alcohol withdrawal (Follesa et al.,
2006; Kumar et al., 2009) (Table 2). The hypothesis of dysregulated
neuroactive steroids in emotional disorders opened a variety of
new targets for treatment. Thus, several researchers worldwide
were stimulated to develop new molecules enhancing neuroster-
oidogenesis and allopregnanolone biosynthesis, in particular to
treat anxiety disorders and depression (Schule et al., 2011).
New hypotheses concerning putative anxiolytic and antidepres-
sant properties of allopregnanolone also involve the role of chronic
stress, hypothalamic-pituitary-adrenocortical (HPA) axis dysregu-
lation and neurogenesis. The importance of allopregnanolone for
the regulation of emotion and its therapeutical use in depression
and anxiety may not only involve GABAergic mechanisms, but
probably also includes enhancement of neurogenesis (Wang et al.,
2005), myelination (Ghoumari et al., 2003), neuroprotection
(Djebaili et al., 2005; Griffin et al., 2004; Mellon et al., 2008;
Sayeed et al., 2006), or regulation of HPA axis function (Reddy,
2006). Acute stress produces an increase in allopregnanolone levels
which negatively modulates the stress-induced HPA axis activation,
thereby facilitating the recovery of physiological homeostasis
following stressful stimuli (Girdler and Klatzkin, 2007; Purdy et al.,
1991). However, repeated and chronic stress leads to a significant
reduction in serum concentrations of allopregnanolone (Dong et al.,
2001; Matsumoto et al., 2005; Serra et al., 2000, 2006). 5a-
reductase, the rate-limiting enzyme in the synthesis of allopreg-
nanolone has been demonstrated to be down-regulated in an
animal model of chronic stress (Agis-Balboa et al., 2007), suggesting
that chronic stress may alter allopregnanolone synthesis leading to
a dysregulation of the HPA axis (Reddy, 2006). Moreover,
allopregnanolone has been demonstrated to restore hippocampal
neurogenesis in a transgenic mouse model of Alzheimer’ disease
(Singh et al., 2012; Wang et al., 2010; Brinton, 2013). In a recent
study in rodents, chronic stress was induced using the social
isolation model (Evans et al., 2012). In this model, chronic stress
was associated with a significant reduction in endogenous
allopregnanolone levels and an enhancement of depressive/
anxiety-like behavior in the novelty-suppressed feeding test and
forced-swim test. Exogenous administration of allopregnanolone
either from the onset of isolation-induced chronic stress or
following a period of chronic stress was able to impede the
occurrence of depression/anxiety-like behavior, to prevent or
normalize HPA axis dysfunction and to avoid impairment of
hippocampal neurogenesis, respectively (Evans et al., 2012).
Interestingly, the behavioral effects of allopregnanolone and its
regulation by antidepressant drugs such as selective serotonin
reuptake inhibitors (SSRIs) are brain region specific (Nelson and
Pinna, 2011). In this animal study, male mice underwent four
weeks of social isolation, thereby inducing dysfunctional behaviors
including fear or aggression in association with a reduction in
allopregnanolone biosynthesis in selected corticolimbic brain
structures. A decrease of allopregnanolone synthesis was found
in the basolateral amygdala, olfactory bulb, hippocampus, and
medial prefrontal cortex but not in the striatum and in the
cerebellum. Moreover, direct infusion of S-norfluoxetine (SSRI) or
pregnanolone (an analog of allopregnanolone) into the basolateral
amygdala, but not into the striatum dose-dependently reduced
aggression in socially isolated mice to a same-sex intruder (Nelson
and Pinna, 2011). These findings suggest an involvement of
allopregnanolone in the regulation of aggressiveness in socially
isolated mice and point to the pivotal role of the basolateral
amygdala and the hippocampus in this context.
The aim of the review is to provide an overview of the animal
and human studies investigating the 3a-reduced neuroactive
steroid allopregnanolone and to discuss its potential in the
treatment of depression and anxiety.
82 C. Schüle et al. / Progress in Neurobiology 113 (2014) 79–87

Table 2
The role of allopregnanolone and other neuroactive steroids in psychiatric and neurological disorders (for details see Reddy, 2010).

Disease Role of allopregnanolone and other NAS Therapeutic implications

Epilepsy Broad-spectrum anticonvulsant properties of 3a-reduced Positive controlled trials for the synthetic neurosteroid
NAS (e.g. allopregnanolone). Sulfated neurosteroids PS and ganaxolone (analog for allopregnanolone) in the treatment
DHEA-S are proconvulsants (blocking of GABAA receptors of epilepsy
and facilitating of NMDA receptor family
Anxiety Anxiolytic properties of progesterone, allopregnanolone, Replacement of neurosteroids by synthetic analogs of
and THDOC. Anxiogenic effects of the sulphated allopregnanolone. Stimulation of endogenous neurosteroid
neurosteorids PS (biphasic in higher dosages) and DHEA-S. synthesis by SSRIs (in this context also called ‘‘SBSSs’’).
In patients with CCK-4 induced panic attacks, there is a Anxiolytic actions of selective ligands for TSPO such as
pronounced decrease in allopregnanolone levels, and FGIN-1-27 or XBD 173
elevated neurosteroids may counteract the occurrence of
spontaneous panic attacks
Premenstrual snydrome (PMS), In normal women, allopregnanolone varies very similarly to Natural progesterone supplementation in women with PMS
pre-menstrual dysphoric progesterone throughout the menstrual cycle with greater has no clear beneficial effect
disorder (PMDD) levels in the luteal phase than in the follicular phase. During
the luteal phase, PMDD women have significantly greater
allopregnanolone levels, coupled with significantly lower
cortisol levels, during both baseline and mental stress
(enhanced ratio of allopregnanolone/cortisol). Marked
insensitivity to benzodiazepine therapy in patients with
PMS (possibly due to cross-tolerance between
benzodiazepines and neurosteroids)
Depression Dysequilibrium of NAS in depressed patients (decreased Increase of 3a-reduced NAS such as allopregnanolone
levels of 3a-reduced NAS Such as allopregnanolone, during treatment with SSRIs, TCAs, and mirtazapine as a
increased concentrations of 3b-THP). Correction of this putative therapeutic principle. Indications, that adjunct
dysequilibrium by antidepressant drugs (SSRIs, TCAs, and DHEA has beneficial effects in depressed patients
mirtazapine). Pregnancy is associated with a marked rise in
progesterone-derived neurosteroid levels, which decline
rapidly after delivery, suggesting a key role of NAS in the
pathophysiology of depression
Learning and memory Neurosteroids such as allopregnanolone regulate both There is preclinical evidence suggesting that
regeneration and repair systems in the brain. In the brain of allopregnanolone may achieve efficacy as a disease
mice with Alzheimer disease, allopregnanolone increases modifying therapeutic to promote regeneration while
expression of liver-X-receptor, pregnane-X-receptor, and 3- simultaneously decreasing the pathology associated with
hydroxy-3-methyl-glutaryl-CoA-reductase (HMG-CoA-R), Alzheimer’s disease
three proteins that regulate cholesterol homeostasis and
clearance from brain. Moreover, allopregnanolone reduces
amyloid-b and microglial activation, and increases markers
of myelin and white matter generation
Alcohol withdrawal Specific GABAA receptor subtypes are sensitive to ethanol at There are new opportunities to intervene in the effects of
doses attained during social drinking while other subtypes ethanol on different aspects of brain function (e.g.
respond to ethanol at doses attained by severe intoxication. inhibition of ethanol effects by blockade of a4-GABAA
Moreover, Furthermore, ethanol increases GABAergic receptors or by inhibition of neuroactive steroid synthesis).
neurotransmission through indirect effects, including the Likewise, it is also possible to enhance ethanol sensitivity
elevation of endogenous GABAergic neuroactive steroids, by targeting a4-GABAA receptors with selective agonists or
presynaptic release of GABA, and dephosphorylation of promoting neuroactive steroid synthesis. The clinical
GABAA receptors promoting increases in GABA sensitivity importance of these findings has to be tested

Abreviations: NAS, neuroactive steroids; PS, pregnenolone sulfate; DHEA-S, dehydroepiandrosterone sulfate; GABAA receptor, gamma-aminobutyric acid type A receptor;
NMDA receptor, N-methyl-D-aspartate receptor; CCK-4, cholecystokinin-tetrapeptide; SSRI, selective serotonin reuptake inhibitor; SBSS, selective brain steroidogenic
stimulant; TCA, tricyclic antidepressant; TSPO, translocator protein (18 kDa); PMS, premenstrual syndrome; PMDD, premenstrual dysphoric disorder; THP,
tetrahydroprogesterone.

2. Allopregnanolone and depression
2.1. Allopregnanolone in animal models of depression
First evidence for the assumption that 3a-reduced neuroactive
steroids may play a role in the pathophysiology of depression came
from an animal study in sham-operated or adrenalectomized/
castrated male rats (Uzunov et al., 1996). In these rats acute
treatment with selective serotonin reuptake inhibitors such as
fluoxetine and to a lesser extent paroxetine increased the brain
levels of allopregnanolone (3a,5a-THP) in a dose- and time-
dependant manner whereas the tricyclic antidepressant imipra-
mine failed to have an influence on the allopregnanolone content
(Uzunov et al., 1996). Interestingly, in this animal study fluoxetine
increased the allopregnanolone biosynthesis without changing the
brain content of its precursor 5a-DHP. Moreover, the administra-
tion of allopregnanolone reduced the duration of immobility in the
forced swim test (FST) in a dose-dependent manner (Khisti et al.,
2000). The same authors could demonstrate that the antidepres-
sant-like effects of fluoxetine but not imipramine in the FST were
mediated by modulation of GABAergic neurotransmission since
the reduction of the immobility caused by fluoxetine was
potentiated by the GABAA agonist muscimol and blocked by the
GABAA antagonist bicuculline, whereas no impact of bicuculline
was found on the antidepressant-like effect of imipramine (Khisti
et al., 2000). In addition, serotonergic agents such as fluoxetine or
fenfluramine have been shown to enhance the antidepressant-like
action of allopregnanolone in the FST by potentiating the
GABAergic tone as a likely consequence of increased brain content
of this neurosteroid (Khisti and Chopde, 2000).
Besides the Porsolt FST, olfactory bulbectomy is one of the most
validated animal models of depression. Bilateral removal of the
olfactory bulbs of rats produces a strong region-specific dysregula-
tion of allopregnanolone homeostasis in the brain which may play
a role in the induction of the bulbectomy syndrome by reducing
the physiological GABAergic tone in particular in the amygdala and
the frontal cortex (Uzunova et al., 2003). Moreover, it has been
demonstrated that this decline of the allopregnanolone content in
 C. Schüle et al. / Progress in Neurobiology 113 (2014) 79–87
selected brain areas can be reversed by sub-chronic (3-week)
treatment with different classes of antidepressants (desipramine,
fluoxetine, sertraline, and venlafaxine) supporting the view that
normalization of allopregnanolone cerebrocortical levels may
contribute to the antidepressant-like profile of these drugs in
the olfactory-bulbectomized rat model of depression (Uzunova
et al., 2004).
Taken together, multiple antidepressant agents (including
fluoxetine, norfluoxetine, fluvoxamine, and paroxetine) (Marx
et al., 2006; Matsumoto et al., 2007; Uzunov et al., 1996), but also
antipsychotic drugs such as olanzapine (Marx et al., 2006) or mood
stabilizers such as carbamazepine (Biggio et al., 1998; Jaworska-
Feil et al., 2000; Serra et al., 2000) have been demonstrated to
elevate allopregnanolone brain levels in animal studies, suggesting
that enhancement of allopregnanolone might be an important
mechanism for the antidepressant effects of these drugs.
2.2. Allopregnanolone and clinical studies in depressed patients
The finding in animal studies that SSRIs such as fluoxetine or
paroxetine increase the content of allopregnanolone in various rat
brain areas (Uzunov et al., 1996) gave reason perform human trials
testing the hypothesis that clinical depression would be associated
with reduced allopregnanolone levels in patients also and
administration of SSRIs would normalize the brain allopregnano-
lone levels in depressed patients thereby improving depressive
symptomatology. Uzunova and coworkers performed a small, but
landmark study in 15 patients suffering from unipolar depression,
measuring the cerebrospinal fluid (CSF) allopregnanolone levels
before and after 8–10 weeks of treatment with fluoxetine or
fluvoxamine (Uzunova et al., 1998). Before initiation of antide-
pressant pharmacotherapy, the allopregnanolone concentrations
were about 60% lower in depressed patients than that determined
in age- and sex-matched non-psychiatric subjects. At the end of the
treatment period, a normalization and re-increase of allopregna-
nolone concentrations in CSF could be observed which was
significantly correlated with the improvement of depressive
symptoms as measured by the Hamilton Rating Scale for
Depression (HRDS). Interestingly, in a recent study in patients
suffering from posttraumatic stress disorder (PTSD), allopregna-
nolone levels in the CSF were decreased being the lowest in those
patients with PTSD and comorbid depression (Rasmusson et al.,
2006). Moreover, a downregulation of allopregnanolone concen-
trations in the CSF could also been shown in this study which was
associated with an increase of PTSD re-experiencing and comorbid
depressive symptoms (Rasmusson et al., 2006).
Consistent with the finding in the CSF in depressed patients, our
research group determined neuroactive steroids in the plasma of
patients suffering from depression, demonstrating that during
depression, the concentrations of 3a-reduced neuroactive steroids
such as allopregnanolone were decreased, while the levels of
isopregnanolone (3b,5a-THP), a stereoisomer of 3a,5a-THP,
which may act as an antagonist for GABAergic steroids (Rupprecht
and Holsboer, 1999) were increased (Romeo et al., 1998).
Moreover, in contrast to preclinical data in these human trials
not only SSRIs (Romeo et al., 1998; Uzunova et al., 1998), but also
other classes of antidepressant drugs, e.g. tricyclic antidepressants
(Romeo et al., 1998), or mirtazapine (Schule et al., 2006b) were able
to correct this disequilibrium of neuroactive steroids throughout
several weeks in depressed patients by enhancing 3a-reduced
neuroactive steroids and decreasing 3b-reduced isopregnanolone
(Romeo et al., 1998; Schule et al., 2006b). However, an association
between amelioration of depressive symptoms (as measured by
the HRDS) and increase of 3a-reduced neuroactive steroids such as
allopregnanolone could only be demonstrated when measuring
neuroactive steroids in the CSF of depressed patients (Uzunova
83
et al., 1998), but not after determination of neuroactive steroid
levels in the plasma (Schule et al., 2006a,b).
In patients suffering from bipolar disorder (BD) or major
depressive disorder (MDD), but in a condition of well-being
without relapse or recurrence during the 3 months before study
enrolment, the plasma concentrations of allopregnanolone and
progesterone were significantly higher than in healthy controls,
also being higher in BD patients than in MDD patients (Hardoy
et al., 2006). The elevated allopregnanolone concentrations did not
depend on concomitant psychopharmacological treatment or
comorbidity with other psychiatric disorder. In contrast to the
described former studies (Romeo et al., 1998; Uzunova et al., 1998)
which were performed with patients during a manifest depressive
episode, the patients of the Hardoy-study were in a state of well-
being which may be the reason for the different results. However, it
has to be mentioned in this context that in another trial, women
with a history of depression, even during full remission, showed
lower concentrations of GABAergic neuroactive steroids such as
allopregnanolone (Girdler et al., 2012).
In two clinical trials our research group investigated the
influence of mood stabilizers such as lithium or carbamazepine on
neuroactive steroid levels in depressed patients (Schule et al.,
2009). In both studies, mirtazapine monotherapy was compared
with combination therapy using mirtazapine and additional
lithium or carbamazepine. In the first study, the mirtazapine-
induced rises in 3a-reduced neuroactive steroids (allopregnano-
lone, pregnanolone) were abolished by additional lithium admin-
istration, as compared to mirtazapine monotherapy. In the second
study, the mirtazapine-evoked increase in allopregnanolone was
reversed after additional administration of carbamazepine also,
suggesting that the mood stabilizers lithium and carbamazepine
do not enhance but rather reverse the increase in plasma
concentrations of 3a-reduced neuroactive steroids in depressed
patients pretreated with antidepressants such as mirtazapine
(Schule et al., 2009).
Investigating depressed patients treated with transcranial
magnetic stimulation (Padberg et al., 2002), sleep deprivation
(Schule et al., 2003), or electroconvulsive therapy (Baghai et al.,
2005), we found no impact of these non-pharmacological
treatment strategies on the levels of neuroactive steroids including
allopregnanolone, indicating that changes in neuroactive steroid
concentrations are not a general therapeutic principle of all kind of
antidepressant treatment, but are rather related to specific
pharmacological properties of antidepressant drugs.
In recent human studies, functional brain imaging was
introduced to further investigate the impact of allopregnanolone
on the regulation of emotion (Sripada et al., 2013; van Wingen
et al., 2008, 2007). In humans, progesterone administration
(increasing downstream allopregnanolone) modulated amygdala
responses to emotional faces (van Wingen et al., 2007) and
enhanced functional connectivity between amygdala and dorsal
medial prefrontal cortex (van Wingen et al., 2008), a regulatory
region interconnected with limbic structures (Price and Drevets,
2010) and crucial to emotional regulation (Phan et al., 2002). In the
trial of Sripada et al. (2013) participants received either 400 mg
pregnenolone (n = 16) or placebo (n = 15). A 3T functional
magnetic resonance imaging was done while performing the
shifted-attention emotion appraisal task to probe emotional
regulation and processing. Compared with placebo, allopregna-
nolone elevations following pregnenolone administration were
associated with reduced activity in the amygdala and insula (all
conditions), but increased activity in the dorsal medial prefrontal
cortex and enhanced connectivity between amygdala and dorsal
medial prefrontal cortex (appraisal condition). Moreover, these
effects on emotion-regulatory neurocircuits were associated with
reduced self-reported anxiety in the 31 healthy male volunteers
84 C. Schüle et al. / Progress in Neurobiology 113 (2014) 79–87
(Sripada et al., 2013). Notably, amygdala and medial prefrontal
cortex are rich in GABAA receptors (Pirker et al., 2000) and
endogenous allopregnanolone (Marx et al., 2006), suggesting that
allopregnanolone could feasibly have a direct influence on the
activity in these brain regions.
3. Allopregnanolone and anxiety
3.1.1. Allopregnanolone in animal models of anxiety
Consistent with their ability to facilitate GABAergic neuro-
transmission, 3a-reduced neuroactive steroids exhibit potent
anxiolytic-like effects in a variety of animal models (Gasior
et al., 1999). Allopregnanolone and pregnanolone produce
anxiolytic-like effects in different animal models of anxiety such
as light/dark transition (Wieland et al., 1995), elevated plus-maze
(Bitran et al., 1991; Rodgers and Johnson, 1998), defensive burying
(Picazo and Fernandez-Guasti, 1995), mirrored chamber (Reddy
and Kulkarni, 1997), and ultrasonic vocalization (Vivian et al.,
1997; Zimmerberg et al., 1994). Thereby, the amygdala was
identified as a key structure for mediating the anxiolytic effects of
allopregnanolone, as direct infusion into the amygdala was
followed by a significant increase in the number of entries and
the time spent in the open arms of the elevated plus maze (Akwa
et al., 1999; Engin and Treit, 2007) or produced antidepressant
effects on the learned helplessness in rats (Shirayama et al., 2011).
Interestingly, allopregnanolone attenuates anxiety-related behav-
ior, but does not affect spontaneous locomotor activity in contrast
to benzodiazepines (Reddy and Kulkarni, 1997; Rodgers and
Johnson, 1998).
In the meanwhile, allopregnanolone has been shown in several
animal studies to reduce stress and anxiety-like behavior in
rodents (Belelli et al., 2005; Darbra and Pallares, 2010; Frye and
Rhodes, 2006; Harrison and Simmonds, 1984; Lambert et al., 2003;
Majewska et al., 1986; Martin-Garcia and Pallares, 2005).
Moreover, blockade of metabolism from progesterone to allo-
pregnanolone impairs social behavior and induces anxiety-related
behavior in rats (Frye and Paris, 2011; Koonce et al., 2012) which
can be reversed by allopregnanolone infusion (Frye et al., 2011).
Decreased corticolimbic allopregnanolone expression during
social isolation may cause increased emotional hyperreactivity,
enhanced contextual fear, and impaired contextual fear extinction
in mice and may serve as a model relevant for posttraumatic stress
disorder (Pibiri et al., 2008). Remarkably, circulating and hippo-
campal concentrations of allopregnanolone but not of corticoste-
rone, progesterone, or estrogen are significantly correlated with
low-anxiety and exploratory behaviors in rats (Koonce et al., 2012).
These results led to the assumption that allopregnanolone may be
one of the most promising candidates within neuroactive steroids
as a new anxiolytic treatment option.
3.2. Allopregnanolone and clinical studies in patients suffering from
anxiety disorders
In humans, no alterations of allopregnanolone concentrations
could be detected in patients suffering from generalized anxiety
disorder (Semeniuk et al., 2001), generalized social phobia
(Heydari and Le Melledo, 2002), or mixed anxiety-depressive
disorder (Bicikova et al., 2000). However, opposite to the findings
in major depression, in patients with panic disorder 3a-reduced
neuroactive steroid levels such as allopregnanolone were in-
creased while the concentrations of 3b-reduced isopregnanolone
were decreased (Strohle et al., 2002). In line with this finding, in
women suffering from panic disorder elevated plasma concentra-
tions of allopregnanolone were found to be elevated both during
the follicular and premenstrual phase of the menstrual cycle
(Brambilla et al., 2003). Thus, it has been hypothesized that
GABAergic 3a-reduced neuroactive steroids may serve as an
endogenous counter-regulatory mechanisms against the occur-
rence of spontaneous panic attacks (Rupprecht, 2003).
No data are available concerning alteration of allopregnanolone
or other neuroactive steroids levels during spontaneous panic
attacks. However, when using infusion with sodium lactate or
cholecystokinin-tetrapeptide (CCK-4) to provoke panic attacks in
an experimental challenge design in patients with panic disorder,
panic symptoms were accompanied by marked decreases in the
3a-reduced allopregnanolone and pregnanolone concentrations,
whereas 3b-reduced isopregnanolone was increased (Ströhle
et al., 2003). In contrast, no such changes in neuroactive steroid
compositions were seen in healthy controls following experimen-
tal panic induction with CCK-4 at dosages of 25 mg i.v. (Ströhle
et al., 2003) or 50 mg i.v. (Zwanzger et al., 2004). Therefore,
alterations of allopregnanolone or other neuroactive steroid levels
do not merely reflect a level of state anxiety but appear to be
related to the pathophysiology of panic attacks in panic disorder
(Ströhle et al., 2003; Zwanzger et al., 2004). Studies investigating
the impact of paroxetine treatment on allopregnanolone in panic
disorder patients revealed conflicting results: In one study,
paroxetine treatment did not affect allopregnanolone levels or
the concentrations of other neuroactive steroids, which were
highly stable over 24 weeks (Strohle et al., 2002). On the contrary,
in another trial the allopregnanolone levels were indistinguishable
between panic disorder patients and healthy controls before
pharmacotherapy; however, 2-month paroxetine treatment in
patients significantly increased the plasma concentrations of
allopregnanolone which were higher than those measured in
controls at the end of the treatment period (Brambilla et al., 2005).
4. Conclusions and future directions
The neuroactive steroid allopregnanolone possesses GABAergic
properties and may have importance for the regulation of emotion
playing an important role in the pathophysiology of affective
disorders such as depression or anxiety. However, the mechanisms
of action of allopregnanolone are not only related to the GABAA
receptor but also include effects on neurogenesis and the
regulation of the HPA system. Studies using animal models and
also trials in patients strongly suggest that allopregnanolone and
other neuroactive steroids are of therapeutic use in the treatment
of affective disorders including depression or anxiety spectrum
diseases. Antidepressant agents, in particular selective serotonin
reuptake inhibitors (SSRIs) such as fluoxetine, paroxetine, sertra-
line have been shown to reverse the decrease of brain allopregna-
nolone in depression or other affective disorders which may at
least in part explain their antidepressant action. Moreover, SSRIs
have been demonstrated to increase allopregnanolone levels and
to correct behavioral deficits expressed by socially isolated mice
even at dosages that are 50fold lower than those required to cause
an effective 5-HT reuptake inhibition (Pinna et al., 2003, 2006).
Therefore, it has been proposed to change the term ‘‘SSRI’’ for these
drugs into the more appropriate term ‘‘SBSS’’ (selective brain
steroidogenic stimulant) (Pinna et al., 2006). However, it remains a
matter of debate if this proposition will become accepted in
psychiatry.
Besides mediation of therapeutic effects of conventional
antidepressant drugs by endogenous metabolites of progesterone,
neuroactive steroids themselves have been used in the treatment of
epilepsy (Herzog, 1995; Reddy, 2011). However, certain pharmaco-
kinetic obstacles limit the therapeutic use of natural neurosteroids.
First, natural neurosteroids such as 3a,5a-THP (allopregnanolone)
have a low bioavailability due to rapid inactivation by glucuronida-
tion and sulphate conjugation. Second, the 3a-hydroxyl group of
 C. Schüle et al. / Progress in Neurobiology 113 (2014) 79–87
allopregnanolone may undergo oxidation to the ketone. Neverthe-
less, synthetic neurosteroids may represent a rational alternative
approach to therapy (Reddy, 2010; Reddy and Kulkarni, 2000). Three
synthetic neurosteroids have been investigated with respect to their
therapeutic potential (ganaxolone, alphaxalone, and minaxolone);
however, until now synthetic neuroactive steroids could not be
established in the treatment of anxiety disorders or depression
(Nothdurfter et al., 2012b).
Regarding endogenous neurosteroidogenesis, recently the
translocator protein (18 kDa) (TSPO) has been identified as a
potential novel target. TSPO is the key element of the mitochon-
drial import machinery facilitating the transport of cholesterol
from the outer to the inner mitochondrial membrane and being
responsible for supplying the substrate cholesterol to the first
steroidogenic enzyme (P450scc), which transforms cholesterol
into pregnenolone, the precursor of all neurosteroids (Papado-
poulos et al., 1997). TSPO ligands (e.g. XBD 173 or etifoxine)
increase neurosteroidogenesis and are a target of novel anxiolytic
drugs producing anxiolytic effects without causing the side effects
normally associated with conventional benzodiazepines such as
sedation or tolerance (Nothdurfter et al., 2012a; Rupprecht et al.,
2009, 2010) (Fig. 2). As such, neuroactive steroids and/or
cholesterol
TSPO ligand TSPO outer
mitochondrial
membrane
inner
mitochondrial
membrane
cholesterol
NEUROSTEROIDOGENESIS
allopregnanolone
Cl-
allopregnanolone
GABAA
receptor
ANXIOLYSIS
Fig. 2. The role of TSPO in anxiolysis (Nothdurfter et al., 2012b). TSPO consists of a
169-amino acid sequence arranged as a five transmembrane helix structure and is
located in the outer mitochondrial membrane. TSPO promotes the transport of
cholesterol to the inner mitochondrial membrane, the rate-limiting step in
neurosteroidogenesis. Within the mitochondrial matrix, cholesterol is converted to
pregnenolone. After diffusion into the cytoplasm, pregnenolone is further processed
into other neurosteroids such as allopregnanolone, which is a positive allosteric
GABAA receptor modulator, thereby exerting anxiolytic efficacy. TSPO ligands, such
as XBD 173 or etifoxine, enhance the transport of cholesterol into the
mitochondrium via TSPO.
85
neurosteroidogenic compounds still are promising avenues for
the treatment of affective disorders such as depression and
anxiety.
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