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The Journal of Neuroscience, July 15, 1998, 18(14):5275-5284
Withdrawal from 3 -OH-5-Pregnan-20-One Using a
Pseudopregnancy Model Alters the Kinetics of Hippocampal
GABAA-Gated Current and Increases the GABAA
Receptor 4 Subunit in Association with Increased Anxiety
Sheryl S.
Smith1,
Qi H.
Gong1,
Xinshe
Li1,
Maria H.
Moran1,
Daniel
Bitran2,
Cheryl A.
Frye3, and
Fu-Chun
Hsu1
1 Department of Neurobiology and Anatomy, Allegheny
University of the Health Sciences, Eastern Pennsylvania Psychiatric
Institute, Philadelphia, Pennsylvania 19129, 2 Department of Psychology, College of the Holy Cross,
Worcester, Massachusetts 01610, and 3 Department of
Psychology, The University of Albany, State University of New York,
Albany, New York 12222
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ABSTRACT |
In the present study, we have characterized properties of steroid
withdrawal using a pseudopregnant rat model. This paradigm results in
increased production of endogenous progesterone from ovarian sources
and as such is a useful physiological model. "Withdrawal" from
progesterone induced by ovariectomy on day 12 of pseudopregnancy resulted in increased anxiety, as determined by a decrease in open arm
entries on the elevated plus maze compared to control rats and
pseudopregnant animals not undergoing withdrawal. Similar findings were
obtained 24 hr after administration of a 5 -reductase blocker to a
pseudopregnant animal, suggesting that it is the GABAA-modulatory 3-OH-5-pregnan-20-one
(3,5-THP) that produces anxiogenic withdrawal symptoms.
Twenty-four hours after steroid withdrawal, the time constant for decay
of GABAA-gated current was also reduced sixfold, assessed
using whole- cell patch-clamp procedures on pyramidal neurons acutely
dissociated from CA1 hippocampus. Thus, 3,5-THP withdrawal
results in a marked decrease in total GABAA current, a
possible mechanism for its anxiogenic, proconvulsant sequelae. In
addition, 3,5-THP withdrawal resulted in insensitivity to the
normally potentiating effect of the benzodiazepine lorazepam (LZM) on
GABAA-gated Cl current. This
withdrawal profile is similar to that reported for other
GABAA-modulatory drugs such as the benzodiazepines (BDZs), barbiturates, and ethanol. These changes were also associated with
significant two and threefold increases in both the mRNA and protein
for the 4 subunit of the GABAA receptor, respectively, in hippocampus. The pseudopregnancy paradigm may be a useful model for
periods of endogenous 3,5-THP withdrawal such as premenstrual syndrome and postpartum or postmenopausal dysphoria, when increased emotional lability and BDZ insensitivity have been reported.
Key words:
GABAA receptor; rat; allopregnanolone; progesterone; -4 subunit; decay time constant; elevated plus maze; kinetics; GABAA-gated current
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INTRODUCTION |
We have previously reported (Gallo
and Smith, 1993 ; Costa et al., 1995; Smith et al., 1998) that the
endogenous steroid progesterone produces withdrawal symptoms via its
GABAA-modulatory metabolite 3-OH-5-pregnan-20-one
(3,5-THP). Upon abrupt discontinuation after chronic
administration of progesterone via injection or implant, increased
anxiety (Gallo and Smith, 1993) and proconvulsant (Smith et al., 1998)
effects are observed. Anxiogenic behavior has been demonstrated after
progesterone withdrawal using light/dark transition (Gallo and Smith,
1993) and the defensive burying paradigm (Gallo and Smith, 1993), as
well as the elevated plus maze (Moran et al., 1996). This anxiogenic
withdrawal property could be prevented by previous administration of a
3-hydroxysteroid oxidoreductase blocker indomethacin (Gallo and
Smith, 1993), suggesting that it is in fact the
GABAA-modulatory 3,5-THP that produces these withdrawal signs. In a similar manner, proconvulsant effects of progesterone withdrawal have been observed (Smith et al., 1998) as
assessed by seizure activity measures: severity, frequency, latency,
and duration after injection of convulsant drugs, picrotoxin, or a
 -carboline benzodiazepine (BDZ) inverse agonist. These withdrawal properties of the GABAA-modulatory 3,5-THP are
similar to those reported for other positive GABAA
modulators, such as the BDZs, barbiturates, and ethanol (Finley and
Nolan, 1989; File, 1990; Kokka et al., 1993).
The GABAA-modulatory 3,5-THP metabolite enhances
GABAA-gated chloride current in hippocampus and spinal
cord, as demonstrated in vitro (Majewska et al., 1986;
Twyman and Macdonald, 1992), and GABAA-mediated inhibition
of cerebellar Purkinje cells assessed in vivo after systemic
administration of progesterone (Smith et al., 1987a,b; Smith, 1989,
1994). Acutely, in vitro application of 3,5-THP
increases binding of the BDZ flunitrazepam to hippocampal and cortical
membranes (Majewska et al., 1986). Chronically administered in cell
cultures at high doses, 3,5-THP can prevent modulation of
GABAA-gated current by BDZs and barbiturates (Roca et al., 1990; Yu and Ticku, 1995). These findings suggest a striking similarity between 3,5-THP and other GABAA-modulatory drugs.
The purpose of the present study was to use a physiological model for
producing sustained elevations in circulating levels of progesterone
and 3,5-THP before steroid withdrawal to characterize anxiogenic
and cross-tolerance withdrawal properties of endogenous neuroactive
steroids. The advantage of the pseudopregnant model is that endogenous
progesterone production by the ovaries is both within physiological
levels and is episodic (Kim and Greenwald, 1986; Robinson et al.,
1981), a condition that may alter the potency of tolerance development
and withdrawal properties, as has been shown for the BDZs (Gallagher et
al., 1986). Withdrawal from 3,5-THP can be induced either by
ovariectomy or by injection of a 5-reductase blocker.
In the present study we tested the hypothesis that steroid withdrawal
produces increased anxiety and changes in sensitivity to BDZ modulation
of GABAA-gated current, assessed electrophysiologically and
behaviorally. In addition, alterations in the kinetics of GABAA-gated current, as well as potential changes in
GABAA receptor subunits relevant for the observed
electrophysiological findings, were investigated after steroid
withdrawal from elevated, circulating progesterone and 3,5-THP
levels produced by pseudopregnancy. This paradigm may be a useful and a
more physiological rat model of both premenstrual and postpartum
conditions than use of injection or implanted capsule.
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MATERIALS AND METHODS |
Subjects
Female Long-Evans rats (Harlan Sprague Dawley, Indianapolis,
IN), 26 d old on arrival, were housed in groups of three under a
reverse light/dark cycle (14/10 hr light/dark). Food and water were
available ad libitum in an environmentally controlled
animal facility. Rats were treated and tested during the dark part of the circadian cycle and were handled only during these times as well.
The stage of the estrous cycle was evaluated by microscopic evaluation
of the vaginal lavage, as described (Smith et al., 1987b).
Hormone and drug administration
Pseudopregnancy. Pregnant mare serum gonadotropin
(Sigma, St. Louis, MO) dissolved in saline was injected
intraperitoneally (20 IU/0.2 ml) on postnatal day 27 (Robinson et al.,
1981; Kim and Greenwald, 1986). Human chorionic gonadotropin (Sigma)
was injected (10 IU/0.2 ml) on postnatal day 29, which was also
considered day 0 of pseudopregnancy. All ovariectomies were conducted
under halothane anesthesia.
Progesterone withdrawal: pseudopregnancy. On day 11 of
pseudopregnancy, the rats were ovariectomized, and ovaries were
weighed. Increased ovarian weight was correlated with elevated serum
3,5-THP levels (r = 0.788) and was considered,
for our purposes, as evidence of increased progesterone release.
(Ovarian weight was increased in 90% of the cases.) In intact,
nonovariectomized rats, serum progesterone and CNS 3,5-THP levels
are well correlated (Corpechot et al., 1993). These rats were tested on
day 12, 24 hr after withdrawal from elevated hormone levels associated
with pseudopregnancy (pseudopregnant wd). Results were compared with
controls, with rats ovariectomized on day 26, and with intact
pseudopregnant animals (day 12).
MK-906 or indomethacin: pseudopregnancy. In some
pseudopregnant groups, rats were given MK-906 (50 mg/kg in oil, i.p.;
Merck, Darmstadt, Germany) on day 11 of pseudopregnancy to inhibit the metabolism of progesterone. Testing on the elevated plus maze occurred
on day 12, as described below, to test the hypothesis that the
withdrawal properties of progesterone were attributable to its
GABAA-modulatory metabolite, as we have previously shown (Gallo and Smith, 1993; Costa et al., 1995; Smith et al., 1998).
Hormone levels. Serum and brain tissue from various
pseudopregnant wd, pseudopregnant, and control groups was collected.
Concentration of the progesterone metabolite 3,5-THP was
determined by RIA (Purdy et al., 1990; Frye et al., 1998) following a
methanol extraction.
Behavioral experiments
Variable speed treadmill locomotion test. Tests were
conducted on a variable speed treadmill (Smith and Chapin, 1996) in a small room lit by an incandescent lamp. The performance of the rats on
the treadmill was recorded with a Beta Max recorder. Each rat was
habituated to the treadmill for 5 min before testing. Rats were scored
for 5 min on their ability to maintain position while locomoting on a
treadmill with speed randomly varied between 4-11 cm/sec. Frequencies
of (1) position slip (slipping back or "riding" the treadmill) and
(2) motor error (bumping into the back of the treadmill) were
determined before and 20 min after an injection of lorazepam (LZM; 0.75 mg/kg, i.p.; Sanofi Co.). A significant increase in either or both of
these parameters was considered a sedative response to the BDZ, which
produces decreased locomotor activity (File, 1981).
Elevated plus maze test. Tests on this apparatus were
conducted in a room with fluorescent lighting and low to minimal noise. The plus maze consists of two open arms, 50 × 10 cm, and two
enclosed arms, 50 × 10 × 40 cm, with an open roof, arranged
so that the two open arms and the two closed arms are opposite each
another. The maze is elevated to a height of 50 cm. On the day of
testing, rats were brought to the room housing the maze. Each rat was
placed at the center of the maze facing an open arm. The frequency and duration of open and closed arm entries were recorded for 5 min by an
observer seated nearby to the maze. An entry into an arm was noted when
the rat placed both forelimbs into the arm. The maze was wiped clean
with 70% ethanol after testing each rat. Anxiogenic effects of
progesterone withdrawal were assessed based on the frequency of open
arm entries versus closed entries as well as the time spent in the open
arms (Pellow and File, 1985; Bitran and Dowd, 1996; Moran et al.,
1996). Decreased time spent on the open arms and a low frequency of
open arm entries relative to control animals is consistent with an
increase in anxious behavior (Pellow and File, 1985).
Electrophysiology
Pyramidal neurons were acutely isolated from CA1 hippocampus 24 hr after ovariectomy or injection of MK-906 (progesterone and/or
3,5-THP withdrawal, respectively). Tissue was digested at 32°C
for 50-60 min under a pure oxygen atmosphere in PIPES-buffered saline
containing (in mM): NaCl 120, KCl 5, CaCl2 1, MgCl2 1, D-glucose 25, PIPES 20, and trypsin
(type XI, Sigma), 0.8 mg/ml, pH 7.0. After a 1 hr enzyme-free
incubation at room temperature, cells were isolated by trituration in 1 ml of 20 mM HEPES-buffered DMEM. The cell suspension was
then plated onto a 35 mm Petri dish, and the cells were allowed to
settle 7-10 min before the DMEM was replaced with recording
medium.
GABAA-activated current was recorded at room temperature
(20-25°C) in a bath solution containing (in mM): NaCl
120, CsCl 5, CaCl2 2, MgCl2 1, TEA Cl 15, 4-aminopyridine 5, HEPES 10, and glucose 25, pH 7.4, 320 mOsm/kg
H2O. The pipette solution contained (in mM):
N-methyl-D-glucamine chloride 120, Cs4 BAPTA 5, and Mg ATP 5. The ATP regeneration system Tris
phosphocreatinine (20 mM) and creatine kinase were added to
minimize GABA rundown.
GABAA-gated Cl currents (10 µM GABA) were recorded in these cells using whole-cell
patch-clamp techniques at a holding potential of  50 mV with a Medical
Systems Axopatch-1D amplifier. GABAA-evoked currents were
filtered at 10 kHz (3 dB, eight-pole low-pass Bessel filter) and
digitally sampled at 500 µsec per point using the pClamp 5.51 software package (Axon Instruments, Foster City, CA). Drug delivery was
accomplished via a solenoid-activated rapid superfusion system
positioned within 50 µm of the cell that released drugs for 20 msec
at 1-3 min intervals to prevent GABA rundown. Peak
GABAA-gated current was calculated as GABA potentiation (or inhibition) for all drug concentrations as (IGABA drug IGABA control)/(I GABA control). Drugs
included LZM, N-methyl--carboline-3-carboxamide (-CC),
RO15-1788, and 3,5-THP (all drugs except RO15-1788 from Research
Biochemicals, Natick, MA).
Kinetics of GABAA-gated current.
GABAA-gated currents (10 µM GABA) were
recorded from acutely isolated CA1 hippocampal pyramidal cells (5-10
msec application) using whole-cell patch-clamp techniques at a holding
potential of 50 mV, as described above. Analysis of current kinetics
was accomplished with the Origin software package (Microcal Software
Inc.) using a pCLAMP transfer module; time constants for rate of
deactivation were determined using a monoexponential decay function and
linear curve fitting. SE was determined using  2
analysis. [This fit was accepted when the sum of squared errors (R2 value) was <0.95]. Current time
course was approximated from the peak current to 90% of recovery.
Western blot analysis
Rats were killed, brains were removed, and hippocampus was
dissected and homogenized, as described (Towbin et al., 1979; Smith et
al., 1998). Hippocampal membranes electrophoresed onto 9% SDS polyacrylamide gels were transferred to a polyvinyl difluoride membrane
(Bio-Rad, Hercules, CA). The 4 subunit of the GABAA receptor was detected with a rabbit antibody (characterized by Smith et
al., 1998) against a peptide [amino acids (aa) 517-523 with an
N-terminal cysteine] of rat 4 as a 67 kDa band (Kern and Sieghart,
1994; Genosys, TX). The  2 subunit of the GABAA receptor
was detected with an antibody (characterized by Smith et al., 1998)
against a peptide (aa 316-352) of rat 2 as a 48 kDa band (Mossier
et al., 1994; Genosys). Negative controls included the use of preimmune
serum (data not shown), as well as blockade with 4 or 2 peptide
(Genosys). Membranes were incubated for 1-2 hr at room temperature
with a 1:5000 dilution of the antibody, followed by a 1:5000 dilution
of horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham,
Arlington Heights, IL). Immunoreactive band densities from the gel for
4 and 2 subunits were quantified using a CCD camera (Umax
Scanner) and one-Dscan software, which uses multiple Gaussian fits to
achieve accurate band density profiles. Results were standardized to a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control protein (36 kDa band).
Semiquantitative reverse transcriptase polymerase
chain reaction
Oligonucleotide primers (Tyndale et al., 1994) for 4 (503 bp)
and 2 (311 bp) subunits of the GABAA receptor (Operon
Inc.) were used with a control primer, GAPDH (657 bp). Total RNA was isolated using Tri reagent and a chloroform extraction and was followed
by reverse transcription (SuperScript II RNase RT, Life Technologies,
Gaithersburg, MD). cDNA containing identical amounts of total RNA was
subjected to PCR amplification at 23-30 cycles (Lau et al., 1993)
using two different concentrations of cDNA (25 or 50 ng). Amplified
cDNA (20-40 µl) was removed for 3.6% NuSieve agarose gel
electrophoresis (FMC Bioproducts, Rockland, ME). Ethidium
bromide-stained DNA bands (single band per PCR product) were calibrated
against a molecular weight marker, and band density was quantified as
described above, standardized to the GAPDH control (Lau et al., 1993).
Values were averaged from both cDNA concentrations at the lower cycle
number known to be in the linear range.
Statistical analysis
Differences between groups were assessed using the Student's
t test (two groups) or standard ANOVA and
Student-Newman-Keuls (>2 groups) post hoc
procedures. In cases where the data did not fit a normal distribution
(>2 groups), the nonparametric procedure, Kruskal-Wallis one-way
ANOVA on ranks, was implemented to determine differences between
experimental and control groups. Significance was determined when
p < 0.05.
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RESULTS |
3,5-THP levels during pseudopregnancy
CNS levels of 3,5-THP assessed on day 12 of pseudopregnancy
(Table 1) were increased to 13.20 ± 0.48 ng/gm, significantly above control values taken from a rat
ovariectomized on postnatal day 26 (1.75 ± 0.06 ng/gm).
Ovariectomy on day 11 of pseudopregnancy reduced 3,5-THP levels
to 3.37 ± 0.7 ng/gm, suggesting that the increase in circulating
levels of this steroid is from an ovarian source. Injection of the
5-reductase blocker MK-906 (50 mg/kg) also significantly reduced
3,5-THP levels to 4.84 ± 0.59 ng/gm. These results suggest
that induction of pseudopregnancy, followed by ovariectomy or injection
of MK-906, is a valid method to produce sustained increased levels of
3,5-THP before withdrawal.
Progesterone withdrawal and the elevated plus maze
Pseudopregnant animals, ovariectomized on day 11 and tested on day
12 (24 hr progesterone withdrawal, pseudopregnant wd), displayed a
decreased amount of time in the open arm of the elevated plus maze
(Table 2; 18 ± 2 sec) compared with
ovariectomized controls (42 ± 3.5 sec) and pseudopregnant animals
(39.8 ± 4.2 sec). In a similar manner, injection of
pseudopregnant rats with the 5-reductase blocker MK-906 on day 11 to
produce 3,5-THP withdrawal also decreased the open arm time when
rats were tested on day 12 of pseudopregnancy (9.8 ± 3.2 sec).
In addition, progesterone withdrawal after ovariectomy resulted in a
significant decrease (p < 0.05) in the number
of open arm entries compared with both pseudopregnant and control
groups (Table 2, 8.8 ± 1.4 pseudopregnant progesterone wd vs
15.6 ± 3.2 pseudopregnant; control, 14 ± 1.7).
MK-906-treated animals also displayed a decreased number of open arm
entries (7.8 ± 2.1; p < 0.01) compared with
controls and pseudopregnant rats. These results suggest that withdrawal
from the 5-reduced metabolites of progesterone increase anxiety-like
behavior in the elevated plus maze paradigm.
GABAA-gated current: progesterone withdrawal
Analysis of the kinetics of GABAA-gated current
recorded using whole- cell patch-clamp procedures in acutely isolated
CA1 hippocampal pyramidal neurons was accomplished using a single order
exponential decay function. The decay time for recovery of
GABAA-gated current to control baseline levels was reduced by a mean 84 ± 9.1% (Fig. 1, Table
3;  = 978.7 ± 114 msec,
pseudopregnant wd vs = 6043.7 ± 1247.3 msec, control;
p < 0.001) after hormone withdrawal in pseudopregnant
rats. Two days after steroid withdrawal the decay time was partially
recovered ( = 3727.8 ± 411 msec) to control values. In
contrast, the decay time constant for GABAA-gated current
was not significantly different from control values when assessed in
pseudopregnant rats not undergoing withdrawal ( = 5879 ± 980 msec, pseudopregnant). The GABA EC50 for peak
GABAA-gated current was unchanged after progesterone
withdrawal, as was the reversal potential for GABA-gated current,
compared to control values (data not shown). The observed decrease in
decay time constant would effectively reduce the total
GABAA-gated current after progesterone withdrawal in
pseudopregnant rats.
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Figure 1.
GABAA-gated current and lorazepam
potentiation: progesterone withdrawal. GABAA (10 µM) current traces before and during application of
lorazepam (LZM, 0.1-10 µM) depict potent
enhancement of GABAA-gated current under control conditions
(CON). In contrast, after progesterone withdrawal
in pseudopregnant animals (PP PWd), LZM was ineffective
in altering GABAA-gated current.
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Benzodiazepine agonists, antagonists, and inverse agonists:
progesterone withdrawal
Twenty-four hours after ovariectomy on day 11 of pseudopregnancy
(progesterone withdrawal), LZM was ineffective in potentiating GABAA-gated current at any dose tested
(108-104 M;
Figs. 1, 2). In contrast, under control
conditions, LZM potentiated GABAA-gated current by 20 ± 0.4 to 102 ± 11.2% across the same concentration range.
However, LZM potentiation of GABAA current was similar to
control values when assessed on day 12 of pseudopregnancy in rats not
undergoing withdrawal: 16.5 ± 4 to 108 ± 10%
potentiation.
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Figure 2.
Progesterone withdrawal and LZM potentiation of
GABAA-gated current: summary diagram. This graph depicts no
significant enhancement of GABAA-gated current by LZM at
any dose after progesterone withdrawal in pseudopregnant animals
(PP PWD) compared to control and pseudopregnant animals
not undergoing withdrawal (PP). In contrast, LZM
significantly potentiated GABAA-gated current by 40-100%
at the highest doses tested
(106-104 M).
n = 15-16 cells per group.
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Under control and pseudopregnant conditions, the benzodiazepine
antagonist RO15-1788 produced only minimal effects on
GABAA-gated current at the highest dose tested (Fig.
3, 8 ± 4.3% at
104 M). In contrast, after
progesterone withdrawal, significant enhancement of
GABAA-gated current by RO15-1788 was observed, from
10.6 ± 5.1% to 58 ± 11.2% across the
108-104 concentration range.
The actions of this antagonist were converted to those of an
agonist.
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Figure 3.
Progesterone withdrawal converts a benzodiazepine
antagonist into an agonist. Graph depicts significant enhancement of
GABAA-gated current by the benzodiazepine antagonist
RO15-1788 after progesterone withdrawal in pseudopregnant animals
(PP PWD). In contrast, under control and pseudopregnant
(PP) conditions, RO15-1788 did not significantly alter
GABAA-gated current. n = 10-12 cells
per group.
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The benzodiazepine inverse agonist, -CC
(108-104 M)
decreased GABAA-gated current by 18.2 ± 2 to
62.1 ± 3% under control conditions (Fig.
4). Similar decreases in
GABAA-gated current were observed after -CC
administration (13 ± 4 to 70 ± 8) to hippocampal
pyramidal neurons from pseudopregnant rats not undergoing withdrawal.
However, after progesterone withdrawal, the ability of -CC to
decrease GABAA-gated current was significantly reduced
(p < 0.05); at this time, inhibition of
GABAA-gated current by 2.3 ± 0.2 to 12.5 ± 3.5% was observed across the
108-104 concentration range
of -CC.
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Figure 4.
Progesterone withdrawal decreases the efficacy of
a benzodiazepine inverse agonist. After progesterone withdrawal
(PP PWD), the inverse agonist -CC was ineffective in
decreasing GABAA-gated current. Under control and
pseudopregnant (PP) conditions, however, this drug was
able to inhibit GABA current by 20-60%. n = 10-12 cells per group.
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3,5-THP potentiation of GABAA-gated current:
progesterone withdrawal
Progesterone withdrawal by ovariectomy on day 11 of
pseudopregnancy resulted in an almost complete inability of the
GABAA-modulatory 3,5-THP metabolite (10 nM) to potentiate GABAA-gated current (Table
4). Under control conditions, this
metabolite increased GABAA-gated current by 81 ± 17.3%. In contrast, after progesterone withdrawal, 3,5-THP
enhanced GABAA-gated current by 11.2 ± 5.2%, an
insignificant level of potentiation.
3,5-THP withdrawal and the 4 subunit of the
GABAA receptor
Because the pharmacological spectrum of BDZ effects as well as the
change in GABAA-gated current decay constant can both be explained by alterations in the 4 subunit of the GABAA
receptor, we tested whether 4 subunit levels were increased after
3,5-THP withdrawal (induced by MK-906) in a pseudopregnant
animal. Western blot analysis (Fig. 5) of
4 band densities revealed a 205.3 ± 12.2% increase in 4
protein levels after 3,5-THP withdrawal (pseudopregnant THP wd)
compared to ovariectomized control values (p < 0.01) after standardization to GAPDH, an invariant protein. In
contrast, no significant change in 2 subunit levels was
observed.
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Figure 5.
3,5-THP withdrawal is associated with
increases in both mRNA and protein for the 4 subunit of the
GABAA receptor. Western blot analysis of 4
(A) and 2 (B) subunit
protein reveals significant threefold increases in 4 (67 kDa band),
but not 2 (48 kDa band) or GAPDH (36 kDa band) levels after
3,5-THP withdrawal in a pseudopregnant rat (PP-THP
Wd) compared to control (con,
p < 0.01). In both cases, staining of the 4 and
2 band could be prevented by previous addition of the appropriate
peptide (con + 4 or 2 peptide).
C, Semiquantitative PCR procedures reveal twofold
increases in 4 mRNA after 3,5-THP withdrawal (PP-THP
Wd) compared to control (con,
p < 0.05). In contrast, no change in GAPDH or 2
mRNA levels were noted. These results are representative of those from
six rats; three replications at two different cycle numbers and
concentrations.
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Assessment of 4 mRNA levels after 3,5-THP withdrawal with
semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) procedures (Fig. 5) revealed a 154.2 ± 9.8% increase in
4 mRNA after 3,5-THP withdrawal (pseudopregnant THP wd) compared to control values (p < 0.05). No
change in GAPDH or 2 mRNA levels was observed across hormone
state.
The sedative potency of LZM: progesterone withdrawal
The sedative potency of LZM was tested using a variable speed
treadmill paradigm (Fig. 6). Under
control conditions, an increase in motor error and position slip is
seen by 20 min after an intraperitoneal injection of LZM (0.75 mg/kg;
"post-LZM increase"). Although LZM was able to significantly
increase both error and slip parameters compared to pre-LZM values
(p < 0.05) after progesterone withdrawal (data
not shown), the percent post-LZM increase was significantly (p < 0.05) reduced (7.2 and 10.3%,
respectively, for error and slip frequencies, progesterone withdrawal)
compared to control values (15.5 and 20.6%, respectively; Fig. 6).
These results suggest that the sedative potency of LZM is reduced by
50% after progesterone withdrawal, compared to its effect under
control conditions.
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Figure 6.
Progesterone withdrawal reduces the sedative
effect of LZM in a variable speed treadmill locomotion task. Increases
in ERROR (bumping into the rear of the treadmill) and
SLIP (slipping back on the treadmill) are observed under
control conditions after administration of 0.75 mg/kg LZM (%
INCREASE POST-LZM) compared to predrug values when rats
are run on a treadmill with varying speed changes. After progesterone
withdrawal (PSEUDOPREGNANT P WD) the post-LZM increases
in ERROR and SLIP were significantly reduced compared with control
conditions, although the sedative effect of LZM was not abolished.
n = 10 animals per group.
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DISCUSSION |
The results from the present study demonstrate that withdrawal
from elevated levels of circulating steroids using a pseudopregnancy paradigm result in a withdrawal syndrome characterized by increased anxiety and cross-tolerance to the benzodiazepines. A previous study
from this laboratory (Smith et al., 1998) demonstrated that progesterone withdrawal also results in increased seizure
susceptibility. These withdrawal effects were shown to be attributable
to abrupt decreases in circulating levels of the
GABAA-modulatory steroid 3,5-THP and were closely
correlated with subsequent increases in CNS levels of the 4 subunit
of the GABAA receptor. As such, this neuroactive steroid
withdrawal syndrome is strikingly similar to withdrawal properties of
other GABAA-modulatory agents, such as the BDZs,
barbiturates, and ethanol (File, 1981; Finley and Nolan, 1989; Kokka et
al., 1993).
The fact that both ovariectomy and administration of the 5-reductase
blocker MK-906 were effective in achieving similar anxiogenic withdrawal effects suggests several conclusions: (1) the greatest source of active steroid pool in this model is the ovary; and (2) a
5-reduced metabolite of progesterone, 3,5-THP, is the active
steroid that triggers withdrawal. These conclusions are not surprising
in view of the fact that this metabolite is a potent positive modulator
of the GABAA receptor, as demonstrated in whole-cell patch
clamp (Majewska et al., 1986), single channel (Twyman and Macdonald,
1992) and in vivo iontophoresis (Smith et al., 1987a,b) studies. The 3,5-THP metabolite increases the duration of opening of the Cl channel in a manner similar to the barbiturates (Twyman and
Macdonald, 1992) and at high doses can increase the frequency of
opening of the Cl channel in a manner similar to the BDZs (Twyman and
Macdonald, 1992). Behaviorally, acute administration of this steroid
directly or indirectly as a result of conversion from systemically
administered progesterone produces anxiolytic (Fernandez-Guasti and
Picazo, 1990; Bitran et al., 1991a,b, 1993; Brot et al., 1997) and
anticonvulsant (Belelli et al., 1989; Devaud et al., 1995; Frye,
1995) effects similar to the GABAA-modulatory BDZs and
barbiturates.
The proconvulsant (Smith et al., 1998) anxiogenic withdrawal properties
of 3,5-THP suggest that increases in neuronal excitability result
from neurosteroid withdrawal. In general, drugs that are anxiogenic and
proconvulsant increase neuronal excitability by a number of mechanisms,
including decreased GABAA response (Zeng et al., 1995), as
well as the development of silent GABAA synapses (Poisbeau
et al., 1997) in hippocampus. In the present study, total
GABAA-gated current was decreased in response to a single 20 msec GABA pulse after progesterone and 3,5-THP withdrawal. This was attributable to a GABAA-gated current with a
markedly faster decay time, compared with control. Thus, on the whole
circuit level, such an effect would be expected to result in a decrease in inhibitory tone with anxiogenic proconvulsant sequelae possible.
In the present study, both electrophysiological and behavioral results
suggest that steroid withdrawal during pseudopregnancy produces
cross-tolerance with the benzodiazepines. The BDZ insensitivity observed here was maximal 24 hr after steroid withdrawal and was not
only a result of chronic progesterone treatment. We have previously reported (Costa et al., 1995; Smith et al., 1998) that withdrawal from
progesterone, administered as continuous release subcutaneous implants,
results in BDZ insensitivity, assessed using whole-cell patch-clamp
techniques on acutely isolated pyramidal neurons from CA1 hippocampus.
This cross-tolerance effect of progesterone withdrawal was again shown
to be attributable to 3,5-THP (Costa et al., 1995; Smith et al.,
1998) and could also be blocked by picrotoxin administration (Costa et
al., 1995), suggesting an action at the level of the GABAA
receptor. A similar BDZ insensitivity has been noted after chronic
treatment and/or withdrawal from BDZs, barbiturates, and ethanol (Buck
and Harris, 1990; Roca et al., 1990).
BDZ insensitivity is observed in women with premenstrual syndrome (PMS)
(Sundstrom et al., 1997), suggesting clinical relevance of these
findings. Altered response to BDZs is also observed across the estrous
cycle or after ovariectomy (Fernandez-Guasti and Picazo, 1990; Bitran
et al., 1991b; Wilson, 1992; Bitran and Dowd, 1996). Additional
evidence for cross-tolerance of BDZs with neuroactive steroids is
provided by behavioral studies, which demonstrate that 3,5-THP
can substitute for several BDZs in a drug discrimination task (Ator et
al., 1993). Other studies have demonstrated that exposure to elevated
levels of 3,5-THP in vitro results in cross-tolerance to the BDZs, as assessed by electrophysiological and biochemical indices (Yu and Ticku, 1995).
In contrast to these similarities, chronic ethanol treatment (Buck and
Harris, 1990) produces certain effects that are opposite to those
observed after progesterone withdrawal in the present study: increased
response to the -carboline inverse agonist and conversion of the BDZ
antagonist RO15-1788 to an inverse agonist. However, progesterone
withdrawal produced a decreased response to a -carboline and
transformed RO15-1788 to an agonist. The reason for the difference in
results is not known, but may be related to one of the many effects of
ethanol on other neurotransmitter systems that could then alter
GABAA and BDZ function. Alternatively, additional
GABAA subunit changes may result from chronic ethanol exposure (Devaud et al., 1997).
The mechanism underlying the withdrawal effects of 3,5-THP
observed in the present study may be related to increases in the
4 subunit of the GABAA receptor. Threefold increases in
levels of the 4 subunit were detected by Western blot analysis in
the present study by 24 hr after steroid withdrawal using the
pseudopregnant model. In addition, significant increases in 4 mRNA
were also observed after 3,5-THP withdrawal, suggesting that one
mechanism for the increase in 4 protein is an effect on
transcription. The pharmacological profile presented here, i.e.,
insensitivity to a BDZ agonist, decreased response to a BDZ inverse
agonist, and conversion of a BDZ antagonist into an agonist is
identical to that reported for oocytes transfected with the 4
subunit of the GABAA receptor (Wisden et al., 1991;
Knoflach et al., 1996; Wafford et al., 1996). Our previous studies
(Smith et al., 1998) have also demonstrated that increases in both mRNA
and protein content of the 4 subunit are observed by 8-24 hr after
withdrawal from progesterone, administered as subcutaneous implants,
for an intermittent 3 week, multiple-withdrawal paradigm.
More importantly, we have also demonstrated (Smith et al., 1998) that
suppression of the 4 subunit by previous administration of 4
antisense, but not missense, oligonucleotides intraventricularly prevents several of the steroid withdrawal properties observed, including (1) the faster decay time for GABAA-gated
current, thus resulting in increased total GABAA-gated
current and decreased neuronal excitability; (2) the BDZ insensitivity,
observed both behaviorally and electrophysiologically; (3) the
alterations in BDZ antagonist effects; and (4) the increased seizure
susceptibility. The sum of these findings suggests that specific
increases in the 4 subunit of the GABAA receptor may
account for the observed withdrawal effects of progesterone and
3,5-THP observed in the present study. Similar increases in 4
mRNA and protein have been described after chronic exposure or
withdrawal from BDZs and ethanol, respectively (Devaud et al., 1997;
Holt et al., 1996; Mahmoudi et al., 1997). Other studies have also
demonstrated altered GABAA-gated current kinetics in
association with altered subunit composition (Lavoie et al.,
1997).
In the present study, steroid withdrawal using the pseudopregnancy
model resulted in tolerance to the GABAA-modulatory actions of 3,5-THP. This type of desensitization has been reported after chronic exposure of cultured cells to high doses of the neurosteroid (Friedman et al., 1993) and across the estrous cycle (Finn and Gee,
1993), but it is not explained by the observed increase in the 4
subunit after steroid withdrawal here. Oocytes transfected with
GABAA receptors containing the 4 subunit do not exhibit a decrease in response to modulation of GABA-gated current by 3,5-THP (Wafford et al., 1996). Other possible mechanisms include (1) altered levels of  or  subunits, both of which have been reported to confer insensitivity to the GABAA-modulatory
actions of this neuroactive steroid (Zhu et al., 1996; Davies et al., 1997), and (2) alteration in phosphorylation state, also reported to result in differential response to GABAA-modulatory
agents (Gyenes et al., 1994).
The use of a pseudopregnancy model here provided several advantages
over use of single or multiple injection or chronic implant, as
reported earlier: (1) it is a more physiological paradigm than the
other procedures, because progesterone is produced by an endogenous, ovarian source; (2) the time course of progesterone exposure, with
progesterone elevated from day 4 to 12, is similar to progesterone exposure during the luteal phase of the menstrual cycle (Dennerstein et
al., 1985; Rapkin et al., 1997), thus making this a useful model for
PMS and postpartum conditions; (3) not only are progesterone and
3,5-THP released, but a number of other steroids are also naturally released by the ovary and corpus luteum, also making this a
better in vivo model (Robinson et al., 1981; Kim and
Greenwald, 1986); (4) release of progesterone under physiological
states, such as estrus, pregnancy, and pseudopregnancy is somewhat
episodic (Robinson et al., 1981; Kim and Greenwald, 1986), a phenomenon that could not easily be replicated by artificial means of
administration. The fact that hormone release is episodic in this model
may explain why a single withdrawal cycle was effective in the present
study in triggering withdrawal properties, whereas in earlier studies, multiple withdrawal cycles were required (Costa et al., 1995; Smith et
al., 1998). Intermittent exposure, as would occur with episodic
release, has been shown to be effective in enhancing withdrawal
properties of ethanol (Kokka et al., 1993) and the BDZs (Gallagher et
al., 1986).
The behavioral data in this study do not reveal as potent a BDZ
insensitivity as the electrophysiological data. This apparent discrepancy may relate to the CNS sites involved. The hippocampus is
valid as a site for spatial memory (Wilson and McNaughton, 1993) and
thus, relevant for the variable speed treadmill task reported here.
However, the sedative effect measured behaviorally may be related to
multiple sites of action throughout the brain.
More importantly, the hippocampus is one relevant limbic structure for
generation of anxiety, as noted by a number of studies indicating that
anxiogenic and anxiolytic effects of BDZ compounds are observed after
local application to the dorsal hippocampus (Huttunen et al., 1986;
Katoaka et al., 1991; Talaenko, 1993). Especially relevant to the
present study is the recent finding that local hippocampal
administration of 3,5-THP produces anxiolytic effects (Bitran et
al., 1997). Other limbic structures, notably the amygdala, are also
well known for their role in anxiety (Britton et al., 1996).
In conclusion, withdrawal from 3,5-THP using the pseudopregnancy
model results in an anxiogenic withdrawal syndrome, characterized by
marked decreases in total GABAA-gated current and
cross-tolerance to the BDZs. These effects were correlated with
significant increases in hippocampal levels of the 4 subunit of the
GABAA receptor, a possible mechanism for the observed
neurosteroid withdrawal effects. Use of the pseudopregnant model here
may have application for increased emotional lability and altered BDZ
response reported for the premenstrual and postpartum periods
(Dennerstein et al., 1985; Sundstrom et al., 1997). In addition,
increased CNS levels of 3,5-THP have been noted during periods of
chronic stress in male rats (Purdy et al., 1991), suggesting that
3,5-THP withdrawal may also arise in association with stressful
episodes in both sexes.
| |
FOOTNOTES |
Received Feb. 9, 1998; revised April 24, 1998; accepted May 4, 1998.
This work was supported by National Institutes of Health Grant DA-09618
to S. S. Smith. The MK-906 was a generous gift from Merck and
Company. We are grateful to R. S. Markowitz for helpful technical
assistance. Correspondence should be addressed to S. S. Smith,
Department of Neurobiology and Anatomy, Allegheny University of the
Health Sciences, EPPI Division, 3200 Henry Avenue, Philadelphia, PA
19129.
| |
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Abstract
Introduction
