Proc. Natl. Acad. Sci. USA
Vol. 93, pp. 12599-12604, October 1996
Fluoxetine-elicited changes in brain neurosteroid content
measured by negative ion mass fragmentography
D. P. UZUNOV*, T. B. COOPERt, E. COSTA*, AND A. GuIDOrrI*
*The Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, Chicago, IL 60612; and tAnalytical Psychopharmacology Laboratory,
Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY 10962
Contributed by E. Costa, July 25, 1996
sham-operated or adrenalectomized/castrated (ADX/CX)
male rats dose-dependently (2.9-58 ,umol/kg i.p.) increased
the brain content of the neurosteroid 3c-hydroxy-5a-
pregnan-20-one (allopregnanolone, 3a,5a-TH PROG). The
increase of brain 3a,5a-TH PROG content elicited by 58
,umol/kg fluoxetine lasted more than 2 hr and the range of its
extent was comparable in sham-operated (--3-10 pmol/g) and
ADX/CX rats (2-9 pmol/g) and was associated with a de-
crease (from 2.8 to 1.1 pmol/g) in the 5a-pregnan-3,20-dione
(5a-dihydroprogesterone, 5a-DH PROG) content. The preg-
nenolone, progesterone, and dehydroepiandrosterone content
failed to change in rats receiving fluoxetine. The extent of
3a,5a-TH PROG accumulation elicited by fluoxetine treat-
ment differed in various brain regions, with the highest
increase occurring in the olfactory bulb. Importantly, fluox-
etine failed to change the 3a,5ca-TH PROG levels in plasma,
which in ADX/CX rats were at least two orders of magnitude
lower than in the brain. Two other serotonin re-uptake
inhibitors, paroxetine and imipramine, in doses equipotent to
those of fluoxetine in inhibiting brain serotonin uptake, were
either significantly less potent than fluoxetine (paroxetine) or
failed to increase (imipramine) 3a,5ci-TH PROG brain con-
tent. The addition of 10 ,uM of 5a-DH PROG to brain slices
of ADX/CX rats preincubated with fluoxetine (10IuM,15
min) elicited an accumulation of 3a,5a-TH PROG greater
than in slices preincubated with vehicle. A fluoxetine stimu-
lation of brain 3a,5ai-TH PROG biosynthesis might be oper-
ative in the anxiolytic and antidysphoric actions of this drug.
Fluoxetine administered intraperitoneally to
Neurosteroids such as 3a-hydroxy-5a-pregnan-20-one (allo-
pregnanolone, 3a,5a-TH PROG); pregnenolone (PREG) sul-
fate; and dehydroepiandrosterone (DHEA), sulfate, promptly
decrease or increase brain excitability acting as potent positive
(3a,5a-TH PROG) or negative (PREG sulfate, DHEA sul-
fate) allosteric modulators of y-aminobutyric acid (GABA)
action at GABAA receptors (1-6). These discoveries have
provided a new mechanism for brain GABAergic tone mod-
ification applicable to the treatment of various neuropsychi-
atric disorder symptoms (7, 8). These include the anxiety and
mood changes of the "late luteal dysphoria syndrome," asso-
ciated with low progesterone (PROG) plasma levels (a steroid
participating in 3a,5a-TH PROG biosynthesis) (8, 9).
Though the prospect that neuroactive steroids could be used in
the symptomatic treatment of specific neurological and psychi-
atric disorders has generated some enthusiasm, substantial diffi-
culties prevent the therapeutic use ofneurosteroids. For example,
the systemic administration of 3a,5a reduced derivatives of
PROG or androstenedione acting as positive allosteric modula-
tors of GABA action at GABAA receptors, indicates that the
doses required to elicit a clear anxiolytic, antidysphoric, and
antiepileptic activity may also produce profound sedation, motor
impairment, or ataxia (6, 8, 10). Two additional pharmacological
actions that may limit the protracted therapeutic use of neuro-
active steroids are: (i) their ability to trigger complex DNA
transcription modifications in neuronal (11) and glial cells (12)
and (ii) a possible tolerance liability, which may limit the pro-
tracted therapeutic use of these compounds in sleep disorders or
convulsive syndromes (13-15).
One might reduce the complications associated with the
protracted administration of neuroactive steroids by develop-
ing drugs that affect selectively some rate-limiting steps of
brain neurosteroid biosynthesis, which unlike that of periph-
eral endocrine tissues, is not under pituitary control (7).
Recently, we were intrigued by a report that fluoxetine's
beneficial effects in the treatment of "late luteal dysphoria"
symptoms occur after a latency time shorter than that de-
scribed for the treatment of the symptoms of depression (16).
Though the mechanism whereby fluoxetine relieves the symp-
toms of late luteal dysphoria remains uncertain, very likely it
may differ from that which alleviates the symptoms of depres-
sion. Since a decrease in brain availability of PROG metabo-
lites may contribute to the onset of late luteal dysphoria
symptoms (9), we began to investigate whether fluoxetine
could change rat brain levels of 3a,5a-TH PROG and its
precursors, including 5a-pregnan-3,20-dione (5a-dihydropro-
gesterone, 5a-DH PROG) and PROG.
We measured simultaneously in the same small brain area
subpicomole amounts of PROG, its Sa metabolites (3a,5a-TH
PROG and 5a-DH PROG), PREG, and DHEA using gas
chromatography negative ion mass fragmentography (GC/NICI-
MF). In fact, steroid radioimmunoassay technology, although
highly reliable for measures of PREG, PROG, and 3a,5a-TH
PROG in thenanomolar range (9,17), loses its intrinsic specificity
and sensitivity in measuring neurosteroids in the picomolar range
(18, 19). The present report, which documents that fluoxetine
increases 3a,5a-TH PROG brain content, also shows that this
action is unrelated to the inhibition of serotonin uptake (20)
elicited by this drug.
MATERIALS AND METHODS
Normal, sham-operated, andADX/CX Sprague-Dawley male
rats (Zivic-Miller), weighing 220-250 g were used. Food and
water or physiologic saline (ADX/CX rats) were available ad
libitum. ADX/CX and sham-operated rats were used for
experiments 10-15 days after surgery. To monitor the results
ofADX, the plasma levels ofcorticosterone were measured by
radioimmunoassay (ICN). All animal procedures employed
were in strict accordance with the National Institutes ofHealth
Abbreviations: PROG, progesterone; PREG, pregnenolone; DHEA,
dehydroepiandrosterone; 3a,5a-TH PROG, 3a-hydroxy-5a-pregnan-
20-one (allopregnanolone); 5a-DH PROG, 5a-pregnan-3,20-dione
(5a-dihydroprogesterone); GABA, y-aminobutyric acid; GC/NICI-
MF, negative ion chemical ionization-mass fragmentographic; ADX/
CX, adrenalectomized/castrated; HFBA, heptafluorobutyric acid an-
hydride; 5,-DH PROG, 5,-pregnane-3,20-dione; GC, gas chromato-
graph; SSRI, selective serotonin reuptake inhibitor.
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
12604Neurobiology: Uzunov et al.
increase 3a,5a TH PROG brain content
The efficacy of inhibitors of serotonin transporter does not correlate with their ability to
3a,5a-TH PROG content,
1.8 + 0.36
720 + 65
7.9 ± 1.3
Compounds were administered in doses of 58 ,umol/kg i.p. 30 min before 3a,5a-TH PROG content
determination and [14C]serotonin uptake analysis. Note that the [14C]serotonin uptake is virtually
completely inhibited (3.4-7.6% ofcontrol) in the group of rats receiving equimolar amounts of fluoxetine,
paroxetine, or imipramine. Data represent the mean ± SEM of 3 to 4 rats.
*P < 0.01 when compared to vehicle-treated rats; **P < 0.05.
serotonergic synaptic activity by selectively blocking its re-
uptake into nerve endings (26). This action may either balance
the equilibrium among different monoaminergic mechanisms
or cause a down-regulation of various serotonergic receptors.
The question we have addressed here is whether the fluox-
etine-elicited increase in brain 3a,5a-TH PROG depends on
a fluoxetine-mediated increase in the amount of serotonin
available to postsynaptic receptors.
The experiment in which the SSRI activity of fluoxetine was
compared with that of equimolar doses of paroxetine (another
potent SSRI) or imipramine (a blocker of serotonin and
norepinephrine reuptake) makes it improbable that serotonin
represents the (still) unknown putative signaling system re-
sponsible for the control of 3a,5a-TH PROG brain biosyn-
thesis. In fact doses of paroxetine and imipramine that, like
those of fluoxetine, completely block serotonin reuptake
(Table 2) produce either a smaller increase (paroxetine) or
even fail to produce an increase of 3a,5a-TH PROG brain
content (imipramine). In view of the fluoxetine and paroxetine
action on the brain content of 3a,5a-TH PROG, however, it
seems likely that 3a,5a-TH PROG may mediate some of the
broad psychopharmacological effects of these two SSRIs, even
though the changes in the level of 3a,5a-TH PROG might not
be correlated with the drugs' effects on the serotonin reuptake.
We thank Dr. E. E. Baulieu (Institut National de la Sante et de la
Recherche Medicale, Paris) and Dr. I. Hanin (Loyola University,
Chicago) for reading our manuscript, and for their helpful suggestions
and constructive critiques. This work was supported by National
Institute of Mental Health Grant RO1 MH49486-04.
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Proc. Natl. Acad. Sci. USA 93(1996)