Departments of 1Physiology and 2Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755
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ABSTRACT |
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Jorge-Rivera, Juan Carlos,
Kerry L. McIntyre, and
Leslie
P. Henderson.
Anabolic Steroids Induce Region- and Subunit-Specific Rapid
Modulation of GABAA Receptor-Mediated Currents in
the Rat Forebrain.
J. Neurophysiol. 83: 3299-3309, 2000.
Anabolic-androgenic steroids (AAS) have become
significant drugs of abuse in recent years with the highest increase
reported in adolescent girls. In spite of the increased use of AAS, the CNS effects of these steroids are poorly understood. We report that in
prepubertal female rats, three commonly abused AAS,
17-methyltestosterone, stanozolol, and nandrolone, induced rapid and
reversible modulation of GABAergic currents in neurons of two brain
regions known to be critical for the expression of reproductive
behaviors: the ventromedial nucleus of the hypothalamus (VMN) and the
medial preoptic area (mPOA). All three AAS significantly enhanced peak synaptic current amplitudes and prolonged synaptic current decays in
neurons of the VMN. Conversely all three AAS significantly diminished
peak current amplitudes of synaptic currents from neurons of the mPOA.
The endogenous neuroactive steroids, 3
-hydroxy-5
-pregnan-20-one and 5
-androstane-3
,17
-diol, potentiated currents in the VMN as
did the AAS. In contrast to the negative modulation induced by AAS in
the mPOA, the endogenous steroids potentiated responses in this region.
To determine the concentration response relationships, modulation by
the AAS, 17
-methyltestosterone (17
-meT), was assessed for
currents evoked by ultrafast perfusion of brief pulses of GABA to
acutely isolated neurons. Half-maximal effects on currents elicited by
1 mM GABA were elicited by submicromolar concentrations of AAS for
neurons from both brain regions. In addition, the efficacy of
10
5 to 10
2 M GABA was significantly
increased by 1 µM 17
-meT. Previous studies have demonstrated a
striking dichotomy in receptor composition between the VMN and the mPOA
with regard to
subunit expression. To determine if the preferential
expression of
2 subunit-containing receptors in the VMN
and of
1 subunit-containing receptors in the mPOA could
account for the region-specific effects of AAS in the two regions,
responses elicited by ultrafast perfusion of GABA to human embryonic
kidney 293 cells transfected with
2,
3,
and
2 or
2,
3, and
1 subunit cDNAs were analyzed. As with native VMN
neurons, positive modulation of GABA responses was elicited for
2
3
2 recombinant receptors,
while negative modulation was induced at
2
3
1 receptors as in the
mPOA. Our data demonstrate that AAS in doses believed to occur in
steroid abusers can induce significant modulation of GABAergic
transmission in brain regions essential for neuroendocrine function. In
addition, the effects of these steroids can vary significantly between
brain regions in a manner that appears to depend on the subunit
composition of GABAA receptors expressed.
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INTRODUCTION |
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Anabolic-androgenic steroids (AAS), synthetic
derivatives of testosterone originally designed to provide enhanced
anabolic potency with negligible androgenic effects (for review,
Kochakian 1993), have become significant drugs of abuse
not only among elite athletes, but among a growing number of
adolescents (Johnson 1990
; Yesalis et al.
1997
) especially young girls (Bahkre et al.
1998
). It has been noted that long term risks from AAS
abuse are greater in women than in men (Franke and Berendonk
1997
; Hickson and Kuowski 1986
; Honor
1997
; Strauss and Yesalis 1993
) and that AAS use
in both women and female rodents is associated with irregular cyclicity (Blasberg et al. 1997
; Bronson 1996
;
Bronson et al. 1996
; Clark et al. 1998a
;
Franke and Berendonk 1997
), accelerated reproductive senescence (Bronson 1996
), and changes in both
aggressive and sexual behaviors (Bronson 1996
;
Bronson et al. 1996
). While adverse effects of long-term
AAS treatment on estrous cyclicity can be attributed to signaling
through androgen receptors (Blasberg et al. 1998
),
recent studies demonstrating that AAS alter Cl
flux in synaptosomes, as well as binding of
t-butylbicyclophosphorothionate (TBPS) and benzodiazepines to
the
-aminobutyric acid type A (GABAA) receptor
(Masonis and McCarthy 1995
; 1996
),
suggest that these compounds may have acute effects in the CNS that are
mediated by nongenomic actions at the GABAA receptor.
Here we show for the first time that three commonly abused AAS,
17-methyltestosterone (17
-meT), stanozolol, and nandrolone, induced rapid modulation of GABAA
receptor-mediated synaptic currents in the ventromedial nucleus of the
hypothalamus (VMN) and the medial preoptic area (mPOA), two forebrain
regions known to play critical, but contrasting, roles in regulating
female reproductive behaviors (for review, McCarthy
1995
). AAS elicited opposing effects in these two
regions, enhancing currents in neurons from the VMN while diminishing
them in neurons from the mPOA. Ultrafast application of GABA plus AAS
to acutely isolated neurons demonstrated that AAS altered GABA efficacy
in a dose-dependent fashion. Moreover, assessment of currents elicited
from recombinant receptors in heterologous cells suggested that the
opposing pattern of AAS modulation in the two brain regions may arise,
at least in part, from the preferential expression of
2 subunit-containing receptors in the VMN and
1 subunit-containing receptors in the mPOA.
Finally, we show that the endogenous neuroactive steroids,
3
-hydroxy-5
-pregnan20-one (allopregnanolone; 3
,5
-THP)
and 5
-androstane-3
,17
-diol (3
-DIOL) also modulated synaptic
currents in both regions; however, AAS and the endogenous neuroactive
steroids had opposite effects in the mPOA, suggesting different
mechanisms of actions for these two classes of steroids at the
GABAA receptor.
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METHODS |
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Animal care and preparation of neuronal tissue
Prepubertal Sprague Dawley female rats [postnatal day PN (3-14)] were employed in this study (n = 245). Animal care procedures were approved by the Institutional Animal Care and Use Committee at Dartmouth and adhere to both the National Institutes of Health and the American Veterinary Medical Association guidelines. For slice recordings, animals were rapidly decapitated, the brains were quickly dissected and placed in ice-cold saline. A thick coronal section was mounted with cyanoacrylic ester (Krazy glue) on the chuck of a Campden Vibroslice microtome (Stoelting, Wood Dale, IL), and 300-µM slices at the level of the VMN or the mPOA were prepared. For isolation of acutely dissociated neurons, the VMN or the mPOA was quickly microdissected from animals of comparable ages. Tissue was minced into small pieces and transferred through several washes and incubated in 0.25% trypsin (Worthington Biochemical, Freehold, NJ) in Opti-MEMI (Gibco Laboratories; Grand Island, NY) at 37°C for 10 min followed by 8-min incubation in 0.2 mg/ml DNase (Sigma Chemical, St. Louis, MO) in the same trypsin-containing medium. Enzyme activity was inhibited by incubation at room temperature in Opti-MEMI containing 5% charcoal-stripped fetal bovine serum (FBS; Gibco) for 8 min. Medium was removed, the tissue triturated, and the cells were plated onto 35-mm tissue-culture dishes coated with Cell-tak (Collaborative Biomedical Products; Bedford, MA). Recordings were made 30-60 min after plating.
Transfection of HEK293 cells
Human 2, rat
3,
rat
2, and human
1
GABAA receptor subunit cDNAs individually
subcloned into the pCDM8 or M13 expression vectors were provided by Dr.
Stefano Vicini (Georgetown University Medical Center, Washington, DC).
Human embryonic kidney (HEK) 293 cells, provided by Dr. Lee Witters
(Dartmouth Medical School, Hanover, NH), were transfected using
Lipofectamine (Gibco) with plasmids expressing cDNAs encoding the
2,
3, and
2 subunits or with ones expressing cDNAs
encoding
2,
3, and
1 subunits (1 µg of each construct).
Cotransfection of the plasmid, pGreenLantern (Gibco), allowed for
selection of transfected cells expressing the green fluorescent protein
(GFP) under fluorescent optics.
Acquisition and analysis of spontaneous inhibitory postsynaptic currents (sIPSCs)
Recordings were made as described previously (Nett et al.
1999) using an Olympus BX50 microscope equipped with a Dage
VE1000 CCD camera system (Optical Analysis, Nashua, NH) from slices
superfused with
95%O2-5%CO2-saturated
artificial CSF (ACSF) containing 125 mM NaCl, 4 mM KCl, 26 mM
NaHCO3, 2 mM CaCl2, 1 mM
MgCl2, and 10 mM glucose supplemented with 10 µM CNQX and 20 µM CPP to block glutamatergic transmission
(Smith et al. 1996
). Pipette saline consisted of 153 mM
CsCl, 1 mM MgCl2, 5 mM EGTA, and 10 mM HEPES to
which 2 mM MgATP was added each day. All chemicals were purchased from
Sigma with the exception of diazepam (RBI; Natick, MA) and nandrolone
(Steraloids; Wilton, NH). Recordings were made at 20-22°C, at a
holding potential (VH) of
80 mV.
Modulators were dissolved in DMSO (0.01% final concentration) and
applied to the bath via gravity flow. Three to 5 min of predrug data
were acquired, the bath was then changed to steroid-containing ACSF (1 µM steroid), and 3-5 min later data were again acquired for 3-5
min. The bath was then switched back to ACSF alone, and 3-5 min were
allowed to pass before postdrug data were collected. Recordings were
made using series resistance compensation of 50-75%. Data were
acquired with a List EPC-7 amplifier (ALA Scientific Instruments;
Westbury, NY) and a PowerMac 8600 and analyzed using HEKA software
(PulseFit; Instrutech; Great Neck, NY) and the Mini Analysis Program
(Jaejin Software; Leonia, NJ.). More than 50 sIPSCs with times to peak <2 ms were acquired, averaged, and analyzed for each drug condition for each neuron, and current averages were fitted under conditions in
which the number of kinetic components, their magnitudes, and absolute
values were not restrained, and the fits were optimized to give root
mean square < 5%. No significant correlations were found
between time to peak versus the value of
1,
time to peak versus the value of
2, the
percentage of the peak current attributed to
1
versus the time to peak, or the value of
1
versus the percentage of the peak current attributed to
1 (linear regression analysis gave multiple
R values and slopes close to 0 for all comparisons) for
sIPSCs recorded from neurons of these two regions.
Recording and analysis of responses from acutely isolated neurons
Responses were elicited by ultrafast perfusion of GABA or GABA
plus steroids for both acutely isolated forebrain neurons and HEK293
cells in the whole cell configuration
(VH = 80 mV) using a LSS-3100
high-speed positioning system (Burleigh Instruments, Fishers, NY) and
solutions described in the preceding text. Assessment of open tip
currents (Lester and Jahr 1992
) indicated that 10-90% of the peak on and off responses that reflect time of solution exchange
were achieved in <500 µs with this system and that applications were
stable with repetitive exposures. Since steroids are known to be
difficult to wash both from cell membranes and from tissue culture
plastic, data were first acquired from separate populations of cells
exposed either to 1 mM GABA alone or exposed simultaneously to 1 mM
GABA plus 1 µM 17
-meT. Subsequent experiments in which the same,
individual cells were first exposed to GABA, then to GABA plus
17
-meT, and finally to GABA alone again (wash) using solution
switching techniques (Zhu and Vicini 1997
) gave
comparable results to those obtained from population studies, and the
data have been pooled. No differences were noted between neurons
exposed to modulator prior to GABA (preequilibrated) and those exposed to GABA and modulator simultaneously. For the experiments presented here, neurons were not preequilibrated with AAS.
Data acquisition and analysis of responses elicited by ultrafast
perfusion to either isolated neurons or to HEK293 cells was made as
described in the preceding text for synaptic currents with the
following modifications. As previously described (Smith et al.
1996), initial assessment of current fits by eye indicated that
the majority of responses were not well fitted by two exponential components with the largest error noted in the fastest component of
current decay as described by
1. Therefore a
least-squares fit using the simplex algorithm was generated for three
exponential components of current decay with no restraints imposed on
any individual component for each elicited response. Plots of all time
constants indicated the presence of three distributions of time
constants and the ranges of values for each distribution. Individual
responses that, when initially fitted with three components, had more
than one time constant that fell within a single distribution were
subsequently refitted with two components. For each cell, time
constants for individual responses were averaged, and these means then
used to determine mean time constants for the populations of cells.
Concentration-response curves were fitted using Prism software
(Graphpad Software, San Diego, CA) using the equation (I = Imax/1 + 10(LogEC50-Log[17
-meT]n) where
Imax equals the current observed with
1 mM GABA alone. Data from both acutely isolated cells and slice
recordings were digitized at 23.6 kHz and filtered at 4 kHz for analysis.
Statistical analysis
Values given are means ± SE. Statistical significance was determined using Kolmogorov-Smirnov test for cumulative distributions, paired and unpaired two-tailed Student's t-tests for assessment of means, and by two-way ANOVA for analysis of concentration response curve fits. All statistical analyses were performed on non- transformed data.
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RESULTS |
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GABAergic spontaneous inhibitory synaptic currents in the VMN and the mPOA
Spontaneous inhibitory postsynaptic currents (sIPSCs) were
recorded in the presence of glutamatergic antagonists (see
METHODS). Responses were reversibly blocked by 10 µM
bicuculline (data not shown), and >50 sIPSCs were averaged per each
cell and each experimental condition. Peak current amplitudes of sIPSCs
from VMN neurons (109.7 ± 6.7 pA) were comparable with those
elicited from mPOA neurons (119.8 ± 10.1 pA). Data collected in
the present study were consistent with previous reports (Nett et
al. 1999; Smith et al. 1996
), indicating that
>90% of individual sIPSCs from the VMN and the mPOA decayed with
biexponential kinetics described by two time constants,
1 and
2 (Table
1). A third component of current decay
(
3 >100 ms) was rarely observed and not
included in the data analysis. In addition, the kinetics of current
decay for sIPSCs were not significantly different from those estimated for miniature IPSCs (mIPSCs; data not shown). All responses
were included in the analysis of sIPSCs with the exception of those with inflections on the rising phase, indicative of asynchronous multiple events, and those events with rise times >2 ms, which may
have been distorted due to cable filtering. Such excluded events
accounted for <10% of those acquired.
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AAS effects on sIPSCs in the VMN and the mPOA
Greater than 60 AAS have been reported to be available on
the United States market (including both the generic and the black markets) (Kammerer 1993). While 17
-meT, stanozolol,
and nandrolone represent three of the most commonly abused AAS
(Kammerer 1993
), the chemical structures of these
compounds represent two structurally distinct groups, the
17
-alkylated derivatives (17
-meT and stanozolol) and the
19-nortestosterone derivatives (nandrolone). In addition to being
chemically distinct from one another, all three AAS have important
structural differences with the neurosteroids. In particular, active
neurosteroids have been reported to possess a 5
- or 5
-reduced steroid ring skeleton with an
-hydroxyl at C3 and a keto group at
either C20 in the pregnane ring or C17 in the androstane ring (Lambert et al. 1995
), structural features that are not
found in any of the three AAS examined here (Fig.
1).
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To determine if AAS could modulate GABAA
receptor-mediated synaptic transmission, neurons in the mPOA and the
VMN 1 µM 17-meT, stanozolol, or nandrolone, a concentration chosen
to approximate that achieved in athletes abusing AAS (Wu
1997
), significantly enhanced the average peak current
amplitude of sIPSCs recorded from neurons of the VMN and significantly
decreased the peak amplitude of sIPSCs recorded from neurons of the
mPOA (Figs. 2 and 6). The fractions of
responsive cells in the VMN were 14/14 (17
-meT), 12/15 (stanozolol),
and 12/14 (nandrolone) and in the mPOA were 11/14 (17
-meT), 13/16
(stanozolol), and 12/14 (nandrolone). Response properties were stable
during the data-acquisition periods prior to, during, and on washout of
AAS, and the effects of each AAS were reversible (Fig. 2B).
In the VMN, 1 µM 17
-meT also acted to enhance GABAergic sIPSCs by
significantly increasing the values of both
1
and
2 without significantly altering the
percentage of the peak current attributable to the fast component
(%
1; Table 1) thus prolonging synaptic
current decay in this region. As with 17
-meT, application of 1 µM
stanozolol or nandrolone significantly increased the values of both
1 and
2 in the VMN
with no change in %
1 (data not shown). In
contrast to the VMN, no significant changes in current kinetics of
sIPSCs for neurons of the mPOA were induced by application of 1 µM
17
-meT (Table 1) or stanozolol (data not shown). In agreement with
results produced by these two 17
-alkylated AAS, no significant
changes in the values of
1 and
2 for mPOA neurons were induced by nandrolone.
However, this 19-nortestosterone derivative did significantly
(P < 0.05) decrease %
1 for
sIPSCs in the mPOA (data not shown). Because the slowest component of
current decay (
3) was only rarely observed in
recordings of sIPSCs, the effects of AAS on this kinetic component of
synaptic responses could not be determined. Finally, AAS did not alter
significantly the frequency of sIPSCs in either region (data not
shown), suggesting the mechanism of action of these compounds is
postsynaptic.
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Responses elicited by ultrafast perfusion of GABA to isolated neurons of the VMN and the mPOA
While analysis of synaptic responses in acutely isolated
slices provides the most physiologically relevant system to assess how
AAS may alter synaptic transmission in the brain, concentrations of
modulators that reach synapses within the slice may be appreciably less
than those in the external solution (Brussaard et al.
1997). To determine the concentration-response relationships
for AAS modulation of GABAA receptor-mediated
currents, experiments were also performed on neurons acutely isolated
from the VMN or the mPOA and exposed to GABA in the absence or presence
of the 17
-meT using ultrafast perfusion techniques.
GABAA receptor-mediated responses (Fig.
3A) were elicited by brief
pulses (3 ms) of 1 mM GABA, application parameters believed to
approximate those in the synaptic cleft (Jones and Westbrook
1995
). Acutely isolated neurons from these postnatal animals
did not adhere tightly to tissue culture substrates, precluding
formation of outside-out patches from these cells with an acceptable
success rate. However, the neurons used for these studies were small
(Cavg = 5.1 ± 0.2 pF for VMN
neurons; n = 164) and 3.8 ± 0.5 pF for mPOA
neurons; n = 152) with estimated diameters between 5 and 10 µm, values comparable with those reported for nucleated
outside-out patches from embryonic mouse forebrain neurons
(Sather et al. 1992
). Responses elicited by brief pulses
of 1 mM GABA were evident in nearly all cells (>90%) and rose rapidly
(10-90% rise times
2 ms), consistent with previous reports
(Nett et al. 1999
; Smith et al. 1996
).
Peak current densities (Ipeak)
elicited by 1 mM GABA from VMN neurons were comparable (648.31 ± 71.66 pA/pF) with those elicited from mPOA neurons (635.76 ± 68.64 pA/pF), and as previously shown (Nett et al. 1999
;
Smith et al. 1996
), the responses in neurons from both
regions were in most cases best fitted by three kinetic components (Fig. 3A; Table 1). Specifically, for neurons from the VMN
(n = 129), current decays for 50% of the
responses were best fitted by three components. For neurons from
the mPOA (n = 112), current decays for 71% of
the responses were best fitted by three components. For both regions,
in those neurons where current decays were best fitted by two
components, it was the fastest component (
1)
that was not observed.
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AAS modulation of responses elicited by ultrafast perfusion of GABA to isolated neurons of the VMN and the mPOA
The AAS, 17-meT, modulated GABAA
receptor-mediated currents from isolated VMN and mPOA neurons in a
manner consistent with its effects on sIPSCs in the two brain regions.
As with sIPSCs, coapplication of 1 µM 17
-meT with 1 mM GABA
significantly increased Ipeak (Figs.
3A and 6) and the value of
1 (Table
1) for neurons of the VMN, but decreased
Ipeak (Figs. 3A and 6) with
no change in
1 or
2
(Table 1) for neurons of the mPOA. Modulation by 17
-meT was elicited
in the absence of preequilibration with the steroid, supporting the
assertion that the AAS act at an extracellular allosteric site on the
receptor. For currents elicited by ultrafast perfusion of GABA to
isolated mPOA neurons, 1 µM 17
-meT also significantly increased
the value of
3 (Table 1), a kinetic component
of current decay rarely observed in synaptic responses. Application of
0.01% DMSO alone, the carrier for AAS, did not elicit responses or
modulate GABAA receptor-mediated currents (data
not shown, n = 5).
While millimolar concentrations of GABA are believed to reflect those
in the synaptic cleft, tonic activation of both synaptic and
extrasynaptic GABAA receptors (Brickley et
al. 1996) may occur at significantly lower concentrations.
Therefore the effects of 1 µM 17
-meT were also assessed for
responses elicited by brief (3 ms) pulses of
10
6 to 10
2 M GABA (Fig.
4A). Measurable responses were
not evident with concentrations of GABA
5 µM, consistent with
previous reports for
2-containing receptors
(Lavoie and Twyman 1996
), and maximal currents were
elicited by 10 mM GABA in both regions. EC50
values for GABA were 32 µM for the VMN and 46 µM for the mPOA, and
the Hill coefficient was ~1 for neurons from both regions.
Coapplication of 1 µM 17
-meT altered the efficacy of GABA,
augmenting Ipeak in VMN neurons and
diminishing Ipeak in mPOA neurons for
concentrations of GABA
10 µM (Fig. 4A). Similar
concentration-response relationships were obtained for the prolongation
of current decay kinetics by 17
-meT in the VMN (data not shown). The
EC50 values for GABA were shifted in response to
coapplication of 1 µM 17
-meT to 11 µM in the VMN and 19 µM in
the mPOA; however, these shifts were not significant.
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To establish the concentration range of AAS required to induce
significant changes of GABAA receptor-mediated
responses under conditions that mimic synaptic transmission,
concentration response relationships were also determined for responses
elicited by brief (3 ms) applications of 1 mM GABA and
108 to 10
4 M 17
-meT
(Fig. 4B). For VMN neurons, significant potentiation of
Ipeak was observed with
10
6 and 10
5 M
17
-meT. Potentiation was not observed for responses from VMN neurons
with 10
4 M 17
-meT. For mPOA neurons,
Ipeak was significantly decreased by
10
6 to 10
4 M 17
-meT
(Fig. 4B). Assessment of concentration response
relationships for neurons from both regions indicated that half-maximal
effects on Ipeak (Fig. 4B)
and decay kinetics (data not shown) were achieved by concentrations of
17
-meT in the submicromolar range: EC50 = 238 nM for the VMN and IC50 = 857 nM for the
mPOA. Application of 17
-meT alone at concentrations between 1-50
µM did not elicit responses in the absence of GABA (n = 10; data not shown).
AAS modulation of recombinant receptors expressed in HEK293 cells
Previous studies have indicated that the predominant isoform of
GABAA receptors expressed in the VMN is
2
3
2
(Wisden et al. 1992
) while the receptor type that
predominates in the mPOA is
2
3
1
(Herbison and Fénelon 1995
; Wisden et al.
1992
). Inclusion of a
1 subunit has
been shown to confer unusual pharmacology for both native
(Bormann and Kettenmann 1988
; Nett et al.
1999
) and recombinant GABAA receptors
(Puia et al. 1991
; Wafford et al. 1993
).
To test if the opposing modulation elicited by AAS in neurons of the
VMN versus the mPOA arises from region-specific expression of
2- versus
1-containing receptors in the VMN and the
mPOA, respectively, we assessed modulation by 17
-meT of GABAergic currents elicited from HEK293 cells transiently transfected with cDNAs
encoding the green fluorescence protein (GFP) and either cDNAs encoding
the
2,
3, and
2 or the
2,
3, and
1 receptor subunits. Previous studies have shown that >90% of cells expressing GFP also expressed GABAA receptors (Zhu et
al. 1996
), a result confirmed in the present study. Recordings
were made from transfected HEK293 cells that were small in size (<5
pF) and not coupled to other cells. Currents elicited by a 3-ms pulse
of 1 mM GABA elicited currents with
Ipeak = 159 ± 47.4 pA/pF for
cells transfected with the
2,
3, and
2 l cDNAs
(n = 5) and 323.6 pA ± 51.5 pA/pF for cells
transfected with the
2,
3, and
1 cDNAs
(n = 7). As with native neurons, current decays of
GABAA receptor-mediated responses from HEK293
cells were described by three time constants as were currents from
native receptors. Decay time constants were
1 = 3.80 ± 0.39 ms,
2 = 67.58 ± 12.54 ms, and
3 = 731.8 ± 211.7 ms for
cells transfected with the
2,
3, and
2 l receptor
subunit cDNAs. Time constants for cells transfected with the
2,
3, and
1 receptor subunit cDNAs were 7.37 ± 2.07, 38.15 ± 10.25, and 228 ± 20.6 ms, respectively, for
1,
2, and
3. Coassembly of a
subunit is known to be
required for benzodiazepine sensitivity of GABAA
receptors (for discussion, Sieghart 1995
). Consistent with inclusion of
, as well as
, and
subunits in recombinant receptors, the benzodiazepine, diazepam, was found to reversibly potentiate Ipeak from both sets of
transfected cells (n = 6; data not shown). Most
important, as with currents elicited from neurons of the VMN, 17
-meT
significantly (P < 0.03; n = 6) and
reversibly enhanced currents from cells transfected with the
2,
3, and
2 receptor cDNAs (Figs. 3B and 6).
Conversely, 17
-meT significantly (P < 0.03;
n = 5) and reversibly decreased currents from cells transfected with the
2,
3, and
1 receptor
cDNAs (Figs. 3B and 6), an effect that mirrors the
modulation by this AAS for neurons from the mPOA. The magnitude of
negative modulation of Ipeak induced in HEK cells transfected with cDNAs encoding the
2,
3, and
1 receptor subunits was not significantly
different from that observed for currents elicited by ultrafast
perfusion of mPOA neurons (Fig. 6). In contrast, while 17
-meT
enhanced current densities for both VMN neurons and HEK cells
transfected with cDNAs encoding the
2,
3, and
2 l receptor
subunits, the magnitude of the increase was significantly higher
(P < 0.001) for the transfected cells (Fig. 6). Taken
together with data from VMN and mPOA neurons, the data from analysis of
responses elicited by ultrafast perfusion of GABA to transiently
transfected HEK293 cells indicate that the opposing pattern of
modulation by AAS can be attributed, at least in part, to the
preferential expression of
1-containing neurons in the mPOA and of
2-containing
neurons in the VMN.
Neurosteroid effects on spontaneous inhibitory synaptic currents in the VMN and the mPOA
While allosteric modulation of GABAA
receptors by endogenous neuroactive steroids is well-characterized (for
review, Lambert et al. 1995), critical
differences in chemical structure between the AAS and the endogenous
neuroactive steroids raised the possibility that these steroid
compounds may have different mechanisms of interacting with the
GABAA receptor. To determine if this was the
case, we also assessed the ability of two endogenous neuroactive steroids, the progesterone derivative, 3
,5
-THP, and the
testosterone derivative, 3
-DIOL, to modulate sIPSCs in neurons in
the VMN and the mPOA. Both 3
,5
-THP and 3
-DIOL potentiated
sIPSCs in the VMN (3
,5
-THP: 5/5 cells; 3
-DIOL: 7/10 cells;
Figs. 5 and 6). However, in contrast to
the diminution produced by AAS in the mPOA, both of these neuroactive
steroids significantly enhanced sIPSCs in the mPOA (3
,5
-THP: 6/10
cells; 3
-DIOL: 7/11 cells; Fig. 5, A and B,
and 6). Testosterone itself, was without effect (Fig. 5B;
n = 5). Enhancement of current amplitudes by endogenous neuroactive steroids and diminution by AAS was observed in the same
neuron in the mPOA (Fig. 5B), indicating that the
differences in responses to AAS and neurosteroids were not attributable
to selective subpopulations of neurons within this region. These data
show that synaptic transmission in forebrain neurons of the VMN and the
mPOA can be modulated by both endogenous neuroactive steroids and AAS
but that the modulation induced by the two classes of steroids for
neurons of the mPOA is not equivalent.
|
Comparison of steroid effects on GABAA receptor-mediated currents
Taken together, our results indicate that all three AAS induced
rapid modulation of GABAA receptor-mediated
currents but that the pattern of AAS modulation differs for neurons of
the VMN versus the mPOA (Fig. 6). Both
the pattern and the extent of modulation in native neurons and HEK
cells is consistent with
2
3
1
receptors predominating in neurons of the mPOA. However, differences in the percent of potentiation between VMN neurons and HEK cells expressing
2-containing receptors (Fig. 6), as
well as previous assessments of subunit mRNA (Wisden et al.
1992
) and protein levels (Fritschy and Mohler
1995
), suggest that receptor subtypes other than
2
3
2
l are also expressed at appreciable levels in this nucleus
and may play a role in determining the sensitivity of VMN neurons to
AAS modulation. Finally, our results indicate that endogenous
neuroactive steroids also modulate GABAA
receptor-mediated currents in neurons from the VMN and the mPOA but
enhance rather than diminish responses in the mPOA, which is in
contrast to AAS in this region (Fig. 6). These data suggest that the
AAS may have different mechanisms for altering
GABAA receptor function than do the endogenous
neuroactive steroids.
|
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DISCUSSION |
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Here we show for the first time that three commonly abused AAS,
17-meT, stanozolol, and nandrolone, can induce rapid and reversible
modulation of GABAA receptor-mediated synaptic
transmission in neurons from the mammalian brain. These data provide
direct evidence that the AAS join a long list of allosteric modulators of the GABAA receptor and suggest a cellular
mechanism by which these compounds may alter CNS function. We found
that peak current amplitudes and decay kinetics of sIPSCs were enhanced
by these three AAS in all responsive neurons of the VMN. In contrast,
peak current amplitudes of sIPSCs were decreased, without concomitant changes in decay kinetics (with the exception of a decrease in %
1 induced by nandrolone), in all responsive
neurons from the mPOA. No significant differences were induced in the
frequency of sIPSCs on application of AAS, suggesting the primary site
of action of these compounds is postsynaptic, a conclusion further supported by the ability of 17
-meT to modulate GABA responses of
isolated neurons. While the endogenous androgen metabolite, 3
-DIOL,
also induced reversible modulation of GABAA
receptor-mediated sIPSCs, application of testosterone did not,
indicating that the effects of these androgens were not due to
nonspecific membrane effects of steroids. Results indicating that
ultrafast perfusion of 1-50 µM 17
-meT in the absence of GABA to
dissociated cells had no effect similarly argue against nonspecific
effects of these steroids.
To obtain concentration response data for AAS under conditions where
both the concentration of GABA and the concentration of the AAS could
be accurately controlled, currents were also elicited from acutely
isolated neurons of the VMN and the mPOA by ultrafast perfusion of GABA
in the absence or presence of AAS. Initial assessment of a single
concentration (1 µM) of 17-meT on responses elicited by brief (3 ms) pulses of 1 mM GABA indicated that this AAS modulated
GABAA receptor-mediated responses from isolated
cells in a manner comparable with that observed for synaptic responses
in intact slices. Specifically, 17
-meT significantly enhanced peak
current amplitudes and the value of
1 for
neurons from the VMN but diminished peak current amplitudes with no
change in
1 or
2 for
neurons of the mPOA. For isolated neurons from the mPOA, 17
-meT also
induce a significant prolongation of
3 of
responses elicited by ultrafast perfusion of GABA. The low frequency of
occurrence of this component in synaptic responses precluded a
comparison of AAS effects on this parameter between the two
experimental paradigms, and the relevance of this component in currents
elicited by ultrafast perfusion remains unclear. It is possible that
there are limitations in solution exchange time with direct perfusion
techniques that arise from the use of the whole cell configuration
(Jonas 1995
), even with the small neurons used in this
study, and although this component was consistently observed, it may
not reflect a physiological process that is relevant to synaptic
transmission in the brain. Conversely, this slow component of decay may
also arise from extrasynaptic receptors that would not be activated
under conditions of low, spontaneous synaptic activity but may
contribute to physiologically relevant GABAA receptor-mediated responses in intact slices under conditions of
high-frequency release and spillover to extrasynaptic receptors (Chéry and DeKoninck 1999
; Rossi and Hamann
1998
). Concentration response relationships indicated that 1 µM 17
-meT significantly altered the efficacy of a broad range of
GABA concentrations (10 µM to 10 mM), suggesting that modulation by
AAS will occur in situ for both saturating (Jones and Westbrook
1995
) and subsaturating (Frerking et al. 1995
;
Hill et al. 1998
; Nusser et al. 1997
)
conditions at neuronal GABAA receptors. AAS may
therefore act to influence not only discrete GABAergic IPSCs but also
the tonic actions of GABA that have been shown influence electrical
excitability (Brickley et al. 1996
).
In the VMN, our data indicate that both AAS and endogenous
neurosteroids potentiated sIPSC amplitudes as well as prolonged the
slower time constant (2) of synaptic current
decay. The observed enhancement of sIPSC amplitude, when taken in
conjunction with data from fast perfusion experiments demonstrating
that concentrations as high as 10 mM GABA were required to elicit
maximal responses from these neurons, suggest GABA concentrations at
synapses in the VMN may be subsaturating as has been reported for other
brain regions (Frerking et al. 1995
; Hill et al.
1998
; Nusser et al. 1997
). Conversely, although
we did not include responses that were notably asynchronous and
multiquantal in our analysis, small inflections in the rising phase may
have not been detected and thus some degree of potentiation of sIPSCs
by steroids may also reflect the integration of multiple mIPSCs whose
durations are prolonged by AAS (Mody et al. 1994
).
With respect to delineating the concentrations of AAS that induce
significant modulation of GABAergic responses, we found that
half-maximal effects of 17-meT were in the hundred nanomolar range
in both the VMN and mPOA. While the physiological range of circulating
endogenous androgens in women has been determined to be appreciably
below this level (2 nM) (Wu 1997
), plasma concentrations of AAS in steroid abusers have been estimated to be 100- to 1,000-fold higher than those of these endogenous androgens (Wu
1997
). Therefore concentrations of AAS that produced
significant modulation of GABAergic currents are likely to be reached
in individuals who abuse these drugs.
GABAA receptors are expressed throughout the
mammalian brain; however, there is a broad range of structural
heterogeneity that in turn gives rise to a plethora of region-specific
differences in GABAA receptor function (for
review, Sieghart 1995). Our results demonstrate that
although the AAS produce significant modulation of GABAergic currents
in both the VMN and the mPOA, these steroids induce an opposing pattern
of modulation in these two brain regions. In conjunction with previous
studies indicating that the VMN and the mPOA differ dramatically in the
expression of
subunit isoforms (Clark et al. 1998b
;
Herbison and Fénelon 1995
; Wisden et al. 1992
; Ymer et al. 1990
), the data presented here
from analysis of recombinant receptors expressed in heterologous cells
strongly suggest that the preferential expression in the mPOA of
receptors containing
1 subunits underlies the
negative modulation of currents in neurons of this region. The
expression of
1-containing receptors is
restricted to a handful of brain regions involved with the production
and affective components of reproductive behaviors (for discussion, see
Nett et al. 1999
), suggesting that the negative allosteric modulation induced by AAS may have unique actions on neuroendocrine functions. While the data from all experiments performed
here are consistent with the predominant expression of
2-containing receptors underlying the positive
modulation by AAS of neurons in the VMN, 17
-meT induced
significantly greater potentiation of responses from HEK cells
transfected with
2,
3, and
2 subunit
cDNAs than from VMN neurons. These data suggest that receptors other
than
2
3
2
combination may contribute to AAS sensitivity. In particular, the role
of
5 (Fritschy and Mohler 1995
;
Wisden et al. 1992
) and
subunits (Davies et
al. 1997
; Whiting et al. 1997
) should be
explored since these receptor subunits are expressed at high levels in
the VMN and have been implicated in determining the actions of other
classes of allosteric modulators (Davies et al. 1997
;
Whiting et al. 1997
; for review, Sieghart 1995
). In addition, our data suggest that receptor
heterogeneity will need to be considered as an important determinant of
how AAS alter GABAA receptor function in brain
regions that play a significant role in mediating aggression and
anxiety, behaviors affected by AAS in both rodents (Bitran et
al. 1993
; Bronson 1996
; Bronson et al.
1996
) and in human abusers (Moss et al. 1992
;
Pope and Katz 1988
).
There is a wealth of data demonstrating that endogenous neuroactive
steroids act as allosteric modulators of GABAA
receptors (for review, Lambert et al. 1995) and that
these compounds have significant effects on neuroendocrine behaviors
(for review, Majewska 1987
). The results presented here,
however, do not support the assertion that the AAS act equivalently to
the endogenous neuroactive steroids in the CNS. Specifically, the
pattern of modulation induced by AAS in the mPOA was significantly
different from that observed with exposure to the endogenous
neuroactive steroids, 3
,5
-THP and 3
-DIOL. All three AAS
induced negative modulation of mean current amplitude in the mPOA,
while 3
,5
-THP and 3
-DIOL enhanced the average peak current in
this region. Moreover, no responses were elicited by concentrations of
AAS as high as 50 µM in the absence of GABA, whereas high
concentrations of neurosteroids are known to activate
GABAA receptors directly (for review,
Lambert et al. 1995
). In fact, the characteristics of
AAS modulation of GABAA receptors in mPOA neurons
are more reminiscent of those reported for the benzodiazepine site
modulator, zolpidem, (Nett et al. 1999
) than to those
elicited by these endogenous steroids. For example, not only do AAS
induce negative modulation in mPOA neurons as does zolpidem, but the
dose-response curve for VMN neurons is bell-shaped as has been reported
previously for both positive and negative modulators acting at the
benzodiazepine site (Rovira and Ben-Ari 1993
;
Sigel et al. 1990
; Stevenson et al.
1995
). It is noteworthy that key structural elements shown to
be common to all active neurosteroids (Fig. 1) (for review, Lambert et al. 1995
) are absent from the three AAS
tested here. While the precise mechanisms by which AAS alter
GABAA receptor function remain to be determined,
our results indicate that AAS may mirror the actions of other
modulators, such as the benzodiazepines, more closely than those
produced by the endogenous neuroactive steroids.
Analysis of AAS and neurosteroid effects on sIPSCs in both the VMN and
the mPOA were performed on animals that were an average age of PN13
(range PN10-PN17). Developmental changes in GABAA receptor subunit composition in the hippocampus have been shown to
result in significant decreases in the sensitivity of granule cells to
the endogenous neurosteroid, 3, 21 dihydroxy-5
-pregnan-20-one (THDOC), between PN10-PN20; a change that may arise from the
late developmental expression of the
subunit (Cooper et al.
1999
; Zhu et al. 1996
). Previous studies have
demonstrated that expression of mRNAs encoding the
l and
2 subunits
(Davis et al., 2000
) as well as the
subunits
(Zhang et al. 1991
) undergo developmental changes in
expression in the VMN and the mPOA during this postnatal period.
Moreover, unpublished data from our laboratory (A. S. Clark, S. Robinson, and L. P. Henderson) indicate that there are significant
changes in expression of the
1 subunit in the
mPOA during the first two postnatal weeks. Both
and
subunits
have been shown to influence the sensitivity of recombinant receptors to 3
,5
-THP (Lambert et al. 1999
; Maitra and
Reynolds 1998
; Puia et al. 1993
), and
subunit expression, as we have shown here, has significant effects on
AAS modulation. Assessment of the percent modulation of sIPSCs as a
function of development for the data in this study suggests that the
magnitude of modulation induced in the VMN and the mPOA by both the AAS
and the neurosteroids may change over this epoch. However, a conclusive
timeline for developmental changes in the sensitivity of neurons in
these regions to steroid modulation will require a more comprehensive
study. In particular, it will be of interest to determine if the
sensitivity to either class of these steroid modulators changes not
only during the period of active synaptogenesis within the first few
weeks of development, but also if there are subsequent changes
concomitant with the onset of puberty.
The ubiquitous expression of GABAA receptors
throughout the brain suggests that AAS will have widespread effects on
a broad range of CNS functions; however, the data presented here have particular relevance with regard to delineating mechanisms by which AAS
may alter reproductive behaviors. For example, GABAergic transmission
in both the mPOA and the VMN regulates the expression of sexual
receptivity in female rodents (for review, Pfaff et al.
1994), and neurosteroid modulators of
GABAA receptors (for review, McCarthy
1995
), including the endogenous androgen, 3
-DIOL, (Frye et al. 1996
) facilitate female sexual behaviors
when acutely infused into the VMN and its efferent targets. Although
chronic AAS exposure may modify sexual behaviors via a number of
signaling pathways, including actions mediated by nuclear androgen
receptors (Blasberg et al. 1998
), our data suggest that
the AAS may also have acute effects on sexual behaviors, as do the
endogenous neuroactive steroids, via actions at the
GABAA receptor. While extrapolation from
receptivity in rats to sexual behaviors in human abusers must be made
with caution, cellular actions of AAS at the
GABAA receptor may underlie some of the reported
changes in sexual performance and libido in steroid abusers
(Franke and Berendonk 1997
). In addition, GABAergic
control of gonadotropin-releasing hormone (GnRH) pulsatility by neurons
of the mPOA is essential both for the onset of puberty (for review,
Ojeda and Urbanski 1994
) and for establishing estrous
cyclicity in adult females (for review, Freeman 1994
).
AAS interactions with GABAergic control neurons of the mPOA may
contribute to the changes in gonadotropin secretion (Bronson et
al. 1996
), irregular cyclicity (Blasberg et al.
1997
; Bronson 1996
; Bronson et al.
1996
; Clark et al. 1998a
), and accelerated reproductive senescence (Bronson 1996
) reported with AAS use.
Although few studies have been carried out on either women or female
rodents, the available data suggest that females are more sensitive to
the actions of AAS than males (Bronson 1996; Hickson and Kurowski 1986
). Previous studies have
demonstrated that AAS effects on benzodiazepine binding to
GABAA receptors are not equivalent in female and
male rats (Masonis and McCarthy 1995
), suggesting that
actions of these steroids at the GABAA receptor
are also sexually dimorphic. Given that the most dramatic increases in
AAS abuse are among women and especially young girls (Bahrke et
al. 1998
), defining how these steroids act in both sexes, as
well as determining if they have significantly different actions in
prepubertal versus adult subjects, will be essential for understanding
the adverse effects of these steroids in the CNS and the implications
their use has for reproductive health.
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ACKNOWLEDGMENTS |
---|
We thank Dr. Stefano Vicini for providing us with GABAA receptor subunit cDNAs and Drs. Ann Clark and Anita Prasad for a critical review of the manuscript.
This research was supported by the National Institute of Neurological Disorders and Stroke (NS-28668 to L. P. Henderson) and the National Science Foundation (DBI-9707826 to J. C. Jorge-Rivera).
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FOOTNOTES |
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Address for reprint requests: L. P. Henderson, Dept. of Physiology, Dartmouth Medical School, Hanover, NH 03755.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 December 1999; accepted in final form 9 March 2000.
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REFERENCES |
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