Department of Physiology, Umeå University, S-901 87 Umeå, Sweden
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ABSTRACT |
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Haage, David and
Staffan Johansson.
Neurosteroid Modulation of Synaptic and GABA-Evoked Currents in
Neurons From the Rat Medial Preoptic Nucleus.
J. Neurophysiol. 82: 143-151, 1999.
The effects of the
neurosteroid 3-hydroxy-5
-pregnane-20-one (allopregnanolone) on
synaptic and GABA-evoked currents in acutely dissociated neurons from
the medial preoptic nucleus of rat were investigated by
perforated-patch recordings under voltage-clamp conditions. The effect
of 2.0 µM allopregnanolone on GABA-evoked currents depended strongly
on the GABA concentration: the currents evoked by 100 µM GABA were
markedly depressed and the desensitization was faster, but the decay
after GABA application was prolonged. In contrast, the currents evoked
by 1.0 µM GABA were markedly potentiated, the activation was faster,
a prominent desensitization was induced, and the decay after GABA
application was prolonged. In the absence of externally applied GABA,
2.0 µM allopregnanolone induced a slow current that could be
attributed to Cl
. Allopregnanolone did not significantly
affect the amplitude of spontaneous tetrodotoxin-insensitive
(miniature) synaptic currents (mIPSCs) originating from synaptic
terminals releasing GABA onto the dissociated neurons. However, the
mIPSC decay phase was dramatically prolonged, with half-maximal effect
at ~50 nM allopregnanolone. A qualitatively similar effect of
allopregnanolone was seen when KCl was used to evoke synchronous GABA
release. The frequency of mIPSCs was also affected, on average
increased 3.5-fold, by 2.0 µM allopregnanolone, suggesting a
presynaptic steroid action.
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INTRODUCTION |
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A number of steroid hormones or their metabolites
have been shown to induce rapid effects on the nervous system. These
effects may occur on a second or sub-second time scale and are thought to be mediated by membrane receptors, in contrast to the more classical, slower genomic action of steroids. Some of the steroids can
be synthesized de novo from cholesterol in the CNS and have been termed
neurosteroids (Baulieu and Schumacher 1996). The perhaps most potent neurosteroid, 3
-hydroxy-5
-pregnane-20-one (also termed 3
,5
-tetrahydroprogesterone or, as below, allopregnanolone) has been shown to dramatically potentiate the membrane currents caused
by
-aminobutyric acidA
(GABAA) receptor activation (Majewska et
al. 1986
; Puia et al. 1990
; Wu et al.
1990
). However, the effects of allopregnanolone on GABA
receptors vary within the nervous system (Gee and Lan
1991
), and with GABA receptor subunit composition (Belelli et al. 1996
; Hauser et al. 1995
,
1996
; Lan et al. 1991
; Shingai et
al. 1991
). The effect of the steroid may also vary with GABA
concentration. Thus allopregnanolone has been reported to potentiate
currents evoked by exogenous application of low (50 µM), but not high
(500 µM), concentrations of GABA onto cultured pituitary cells
(Poisbeau et al. 1997
). Similar results have also been
reported for pregnanolone, a 5
-stereoisomer of allopregnanolone (Le Foll et al. 1997
).
The dependence on GABA concentration raises the question whether the
steroids affect GABAA receptors activated by
synaptically released GABA, which is likely to reach near-millimolar
concentrations (see, e.g., Edwards 1995). Recent results
on GABA-mediated synaptic currents have not provided a unified answer.
Thus no effect on the peak amplitude, but a prolonged decay (which
depended on subunit composition) of synaptic currents was reported by
Brussaard et al. (1997)
(see also Harrison et al.
1987
). Similar effects have been reported for pregnanolone
(Reith and Sillar 1997
). However, in the study by
Poisbeau et al. (1997)
, although presynaptic effects were suggested, miniature synaptic currents were not affected by
allopregnanolone, and thus no evidence was found in favor of a
postsynaptic action of allopregnanolone during synaptic transmission.
The present study concerns neurons from the preoptic area of rat. The
preoptic area is involved in the regulation of sexually related
functions, as well as in thermoregulation, slow-wave sleep, and
feeding. The majority of gonadotropin releasing hormone
(GnRH)-producing cells are located in the preoptic area (see, e.g.,
Chappel 1985). The involvement of GABA in the regulation
of GnRH release has been clearly shown (Leonhardt et al.
1995
), and feedback by gonadal steroid hormones is suggested to
affect GABA-mediated transmission in the preoptic area (Grattan
et al. 1996
; Herbison 1997
). However, fast
steroid effects on membrane currents of preoptic neurons are, to our
knowledge, not known, although it has been demonstrated that medial
preoptic neurons can generate currents mediated by
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid,
N-methyl-D-aspartate, GABAA, and glycine receptors (Karlsson et
al. 1997a
,b
).
The present work was carried out to investigate the effect of the
neurosteroid allopregnanolone on GABAA
receptor-mediated currents in neurons from the rat medial preoptic
nucleus (MPN). The aim was to characterize the neurosteroid effects on
the currents induced by synaptically released, as well as externally
applied, GABA. We used a preparation of acutely dissociated medial
preoptic neurons, with adherent functional synaptic terminals
co-isolated with the neurons (Haage et al. 1998). We
report that allopregnanolone affects the time course of GABA-evoked
current in a manner that depends on the GABA concentration and results
in either depression or potentiation. We further report that the main
effect of allopregnanolone on synaptically evoked currents is to
prolong the time course of current decay. In some cases
allopregnanolone also causes an increased frequency of spontaneous
transmitter release. Some of the results have been reported in a
preliminary form (Johansson and Haage 1997
).
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METHODS |
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Cell preparation
The cells were prepared from hypothalamic brain slices from
young male Sprague-Dawley rats, 50-120 g in weight. Ethical approval was given by the local ethics committee for animal research. The procedures for preparation of slices and for mechanical dissociation of
cells have been previously described (Johansson et al.
1995; Karlsson et al. 1997b
). In short, after
decapitation without use of anesthetics, the brain was removed and
placed in preoxygenated ice-cold incubation solution (see
Solutions) for ~5-10 min. A block of tissue
containing the preoptic area and anterior hypothalamus was cut out, and
coronal slices, 250-300 µm thick, were cut with a vibroslicer (752 M, Campden Instruments, Leicestershire, UK). The slices were
incubated for at least 1 h in incubation solution, at 29-31°C
or at room temperature (21-23°C), before isolation of cells. The
mechanical vibrodissociation method by Vorobjev (1991)
was used after slight modification (cf. Pasternack et al. 1993
), and without enzymes. The mechanical vibration was
applied by a glass rod (~0.5 mm diam), mounted on a piezo-electric
bimorph crystal and directed to the site of the medial preoptic
nucleus. The dissociated cells were allowed to settle at the bottom of a Petri dish for ~10 min. The cell bodies were 10-15 µm at their longest axis, rounded or elongated in shape, typically with two or more
5- to 30-µm-long dendritic processes, but in some cases with longer
neurites or without neurites. In a majority of cells, spontaneous
synaptic currents indicated the presence of co-isolated functional
synaptic terminals (see RESULTS) (Haage et al.
1998
).
Electrophysiological recordings
Whole cell patch-clamp (Marty and Neher 1983)
recordings were performed by the use of the amphotericin-B
perforated-patch technique (Rae et al. 1991
). All
currents were recorded under voltage-clamp conditions. Borosilicate
glass pipettes (GC150, Clark Electromedical Instruments, Pangbourne,
UK), with a resistance of 2-7 M
when filled with intracellular
solution and immersed in extracellular solution (see
Solutions), were used. The liquid-junction potential
between pipette solution and extracellular solution was measured as
described by Neher (1992)
, and has been subtracted in
all potential values given. The signals were recorded using an Axopatch
200A amplifier, a Digidata 1200 interface, and pClamp software (all
from Axon Instruments, Foster City, CA) controlled via a 486-processor
based personal computer. Recorded signals were low-pass filtered at
2-10 kHz (
3 dB). Series-resistance compensation was not used, due to
its introduction of extra noise. However, to avoid experiments with
large changes in series resistance, and to evaluate patch integrity,
the time course of capacitative current elicited by a
5-mV voltage
step was repeatedly monitored during the experiments. Rare occasions of
sudden changes in capacitative current resulting in a faster current
decay were followed by a gradual increase of the noise level, decrease
in membrane resistance, and usually diminished GABA-mediated currents.
This was interpreted as patch rupture followed by amphotericin
B-mediated effects on cellular membranes. The latter recordings were
therefore discarded.
In a majority of experiments, the currents were recorded at a constant
membrane potential of 34 mV. At this potential, the driving force for
Cl
-mediated currents was reasonably large,
whereas additional noise and instability of membrane properties
appearing at more positive potentials were avoided.
All solutions, for continuous perfusion as well as for application of test substances, were applied by a gravity-fed fast perfusion system, controlled by solenoid valves operated from the computer. The solution exchange time, as indicated by the current change measured from a patch pipette in alternating extracellular solution and 140 mM KCl, was in good cases <10 ms. All experiments were performed at room temperature (21-23°C).
Solutions
The incubation solution used during the preparation and for
storage of slices contained (in mM) 150 NaCl, 5.0 KCl, 2.0 CaCl2, 10 HEPES, 10 glucose, and 4.93 Tris-base.
This solution was used supplemented with a gas mixture containing 95%
O2-5% CO2. The extracellular solution used during recording of currents contained (in
mM) 137 NaCl, 5.0 KCl, 1.0 CaCl2, 10 HEPES, and
10 glucose. Glycine (3 µM) and tetrodotoxin (2 µM; Sigma, St.
Louis, MO, or Alomone Labs, Jerusalem, Israel) were routinely added,
and pH adjusted to 7.4 with NaOH. When used (see RESULTS),
GABA, allopregnanolone (3-hydroxy-5
-pregnane-20-one), or
bicuculline methiodide (all from Sigma) was added to the extracellular
solution. The steroid was dissolved in ethanol (max ethanol
concentration 0.01%). Control experiments without steroid revealed
that ethanol did not significantly affect the recorded parameters. In
some experiments, bovine serum albumin (0.01%, wt/vol) was also added,
without causing any significant difference in recorded parameters. The
intracellular solution, used for filling the pipette, contained (in mM)
140 K-gluconate, 3.0 NaCl, 1.2 MgCl2, 1.0 EGTA,
and 10 HEPES; pH was adjusted to 7.2 with KOH. Amphotericin B (Sigma),
prepared from a stock solution (6 mg amphotericin B dissolved in 100 µl dimethylsulphoxide), was added to a final concentration of 120 µg amphotericin B per ml intracellular solution.
Analysis
The analysis was performed by the use of pClamp software (see
Electrophysiological recordings) and Origin software
(version 4.0-4.1, Microcal Software, Northampton, MA). Miniature
synaptic currents were detected by visual inspection, and the amplitude and time course of each event were measured semi-manually by using cursors and the curve fitting routines provided by the software. The
lower limit for detection was ~5 pA. For estimating the number of
exponential components, the standard deviation of the difference between recorded data and the theoretical curve obtained by a simplex
fitting algorithm was compared. When this standard deviation was
reduced <2.0% by an additional component, the latter was not accepted. The same criterion was used for evaluating the time course of
current evoked by external GABA application. In all cases, for
miniature inhibitory postsynaptic current (mIPSC) decay, GABA-evoked
desensitization and deactivation, a majority of events were best
described by a single exponential function. The steady leak current has
been subtracted in the figures. The continuous line (dose-response
curve) in Fig. 4B, relating mIPSC decay time constant ()
to the allopregnanolone concentration (C), is described by
the equation
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(1) |
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RESULTS |
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The results presented below were obtained from 93 medial preoptic
neurons. As previously described, all neurons tested respond to
application of 100 µM to 1 mM GABA with currents that can be attributed to the activation of GABAA receptors
(Karlsson et al. 1997a). The currents evoked by GABA
show desensitizing components that recover only slowly (time constant
5-10 s) after wash out of GABA. In the present study, therefore, GABA
was applied (for 0.32-2.56 s) at intervals of 20-40 s. This resulted
in similar responses at repeated applications.
Effects of perfusion with allopregnanolone on responses to 100 µM GABA
The current responses to application of 100 µM GABA
were recorded at a holding membrane potential of 34 mV. We used 100 µM GABA because this is probably near the concentration reached in central synapses (see Edwards 1995
, for review). When
GABA was applied, an outward current that reached a peak of 209 ± 34 pA (mean ± SE; 10-90% rise time 111 ± 11 ms;
n = 21) was generated (Fig.
1A). The subsequent
desensitization in the presence of GABA was examined in 13 cells to
which GABA was applied for 2.56 s. The desensitization was best
described by a single exponential function (see METHODS),
with a time constant (
desens) of 1.7 ± 0.1 s in 10 of the 13 cells. In the remaining three cells, the time constant was too long to be estimated from the 2.56 s of GABA
application. The current amplitude after 2.56 s was 52 ± 4%
(n = 13) of the peak amplitude. After the end of the
GABA application, the current decay (deactivation) to baseline was also
best described by a single exponential function, with a time constant
(
off) of 694 ± 70 ms (n = 21).
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The effect of the steroid allopregnanolone (2.0 µM) was tested after
a minimum of 30-s perfusion with allopregnanolone-containing extracellular solution and subsequent application of a test solution that contained 100 µM GABA as well as 2.0 µM allopregnanolone. The
evoked current now was markedly depressed (to 54 ± 5% at peak, n = 21) compared with the control situation described
above (Fig. 1A). Further, the desensitization was faster in
allopregnanolone (time constant, desens,
1.1 ± 0.1 s; n = 10), although the fraction of the peak amplitude reached after 2.56 s of GABA application was
not significantly affected. The decay to baseline current after the end
of the test application was, in 20 of 21 cells, prolonged. The time
constant (
off) was 1,541 ± 205 ms in the 21 cells; in 10 of the 21 cells the time constant was more than twice
as long as in control solution. The relative effects of allopregnanolone on different current parameters are summarized in Fig.
1B. The effects were reversible within 1 min of wash with control solution.
From above, it is thus clear that in MPN neurons allopregnanolone reduces the current during application of 100 µM GABA but prolongs the decay phase after GABA application.
Effects of perfusion with allopregnanolone on responses to 1 µM GABA
In several previous studies reporting steroid-induced potentiation
of GABA responses, lower concentrations of GABA have been used
(Hauser et al. 1995; Kokate et al. 1994
;
Shingai et al. 1991
; Wu et al. 1990
;
Zhang and Jackson 1994
). Although the GABA concentration used above in the present study was chosen to be in the
near-physiological range, we considered the differences in
concentration as a possible reason for the discrepancy between the
results above and earlier reports (see also INTRODUCTION).
We therefore investigated the responses to application of 1.0 µM
GABA. When 1.0 µM GABA was applied in control solution (standard
extracellular solution, see METHODS) at
34 mV, a slowly
rising outward current was generated (10-90% rise time, 1,162 ± 150 ms; n = 9), reaching a relatively steady level of
32 ± 8 pA (n = 9). No or a very weak
desensitization was seen during a standard application of 2.56 s
duration, nor (as tested in 1 cell) during longer applications of
nearly 20 s duration. The return to baseline after the end of the
GABA application was monoexponential with a time constant of 398 ± 63 ms (n = 9).
The effect of allopregnanolone was studied as above, after a minimum of
30 s perfusion with allopregnanolone-containing solution. When 1.0 µM GABA was applied in the presence of 2.0 µM allopregnanolone, the
results differed dramatically from those described above. The
GABA-evoked current was markedly potentiated (201 ± 28% at peak,
n = 9) compared with control. The onset of current was
more rapid with a distinct peak reached after a rise time (10-90%) of
291 ± 30 ms (n = 9) (Fig. 1C). Thus a
prominent desensitization was seen, to 66 ± 6.7% of peak
amplitude after 2.56 s of GABA application (n = 9). The time constant of return to baseline after the GABA application,
off, was prolonged to 707 ± 59 ms
(n = 9). The relative effects of allopregnanolone are
summarized in Fig. 1D. It was thus clear that the effect of
allopregnanolone depended dramatically on the GABA concentration, with
a reduction of peak current at 100 µM GABA and a potentiation at 1.0 µM GABA.
Direct effects of allopregnanolone
The results above describe the effects of allopregnanolone on
GABA-evoked currents. However, the steroid by itself induced a membrane
current, in the absence of applied GABA. Thus, when 2.0 µM
allopregnanolone was applied at 34 mV, an outward current was
generated (Fig. 2, A and
B). The current showed a transient component, with a peak of
32 ± 6 pA (n = 10) reached within 0.2-4.3 s, and
a subsequent decay to a steady component of 0-27 pA. (Half-peak amplitude was reached after 10-35 s in 8 cells, but was not reached within 35 s in 2 cells.) The relation between peak current and membrane potential (Fig. 2C,
) showed some outward
rectification, and a reversal potential near
74 mV (2 cells), in
similarity with the currents evoked by GABA (Karlsson et al.
1997a
). When a high Cl
concentration
was used in the pipette solution (140 mM KCl substituted for
K-gluconate), the reversal potential was 0 ± 2 mV
(n = 3), as expected from a Cl
current (Fig. 2C,
). The current evoked by
allopregnanolone was completely, but reversibly, blocked when the
GABAA receptor blocker bicuculline methiodide
(100 µM) was added to the extracellular solution (n = 3; not shown).
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The direct effect of allopregnanolone described here may be of importance for interpreting the reduction of GABA-evoked currents described above. It seems possible that part of the allopregnanolone-induced reduction of current in response to 100 µM GABA is due to a number of receptors already being activated or desensitized by allopregnanolone itself (i.e., before GABA application), thus reducing the channel population available for contributing to the response on GABA application. However, the peak current directly evoked by 2.0 µM allopregnanolone (32 ± 6 pA; n = 10) was considerably smaller than the reduction (96 ± 22 pA; n = 21) of the peak response to 100 µM GABA caused by allopregnanolone. Similarly, when compared in the same cell, the current evoked by allopregnanolone showed a peak amplitude that constituted only 29 ± 7% (n = 4) of the reduction of current evoked by 100 µM GABA. Thus the number of channels contributing to the peak response caused by allopregnanolone in the absence of GABA is not sufficient to account for the reduction of current activated by GABA.
Effects of allopregnanolone on spontaneous synaptic currents
From above, it is clear that the effect of allopregnanolone
depends on the GABA concentration. It is thus important, for evaluating the physiological role of steroids in the nervous system, to
investigate the effects on synaptically released GABA in physiological
concentrations. For this purpose, we took advantage of the synaptic
boutons that are easily co-isolated with the medial preoptic neurons.
These synaptic boutons spontaneously release GABA and thereby give rise to tetrodotoxin-insensitive mIPSCs (Haage et al. 1998).
When such mIPSCs were recorded in control solution, at a holding
potential of
34 mV (59 cells), they showed a rapid (usually <2 ms)
rise phase followed by a monoexponential or doubly exponential decay. The amplitude distribution deviated from a Gaussian, being skewed to
the right (Fig. 3A). The peak
occurred at 17 pA, and the mean amplitude was 26 pA (±1 pA,
n = 1,470; range of mean 9-46 pA for 34 individual
cells). In the presence of 2.0 µM allopregnanolone, the mIPSC
amplitude distribution (mean amplitude 27 ± 1 pA;
n = 1,298) was not significantly different from control
(Fig. 3B).
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The decay of the mIPSCs was best described by a single exponential function in a majority of cases (64% of 56 events from 29 different cells used for an initial evaluation). Although some mIPSCs were better described by two exponential components according to the criterion used (see METHODS), the improvement was not marked as judged by visual inspection. Thus, to simplify the comparison with mIPSCs recorded in the presence of the steroid (when also ~64% of mIPSCs showed a monoexponential decay), the decay is for all events described in terms of best fitted single exponential function. In control solution, the time constant of the mIPSC decay was 19.1 ± 0.4 ms (599 events pooled from 31 cells; range of mean 8-31 ms for individual cells). In the presence of 2.0 µM allopregnanolone, the time course of decay was considerably prolonged (Fig. 4, A-D): mean time constant was 133 ± 4 ms (n = 165; range of mean 90-181 ms for 15 individual cells).
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We investigated the relation between allopregnanolone concentration and the effect on the mIPSC decay. A slight prolongation of the mIPSC decay was observed already at concentrations of 6.3-20 nM, and a near-maximal effect at >200 nM allopregnanolone. Although the effect was less at 2.0 µM than at 200 nM allopregnanolone, thus suggesting a more complex interaction, the data are shown together with a curve described by Eq. 1 (see METHODS) for a comparison. The concentration for half-maximal effect was ~50 nM (Fig. 4B).
Effects of allopregnanolone on mIPSC frequency
In many cases, perfusion with allopregnanolone (200 nM to 2.0 µM) resulted in an increased frequency of mIPSCs (Fig. 4A), thus suggesting a presynaptic steroid action. The effect was evaluated by comparing the mIPSC frequency during 30-s intervals immediately before and immediately after the onset of allopregnanolone perfusion. Although there was a prominent effect in some cases (Figs. 4A and 5A), there was a large variability between repeated allopregnanolone applications in individual cells and also a large variability between cells. The overall effect of 2.0 µM allopregnanolone was to increase the mIPSC frequency by a factor of 3.5 ± 0.8 (n = 36: number of cells). Although not evaluated quantitatively, the effect was often more prominent when there was a low mIPSC frequency in control solution (Fig. 5A), whereas with high control frequency, allopregnanolone sometimes reduced the mIPSC frequency (Fig. 5B).
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Effects of allopregnanolone on KCl-evoked synaptic currents
Because the effects of allopregnanolone depended on GABA
concentration and included changes in current kinetics, it seemed possible that the effects on evoked transmitter release might differ
from the effects on the spontaneous mIPSCs. The effect on mIPSC
frequency also suggested that the release probability was affected,
which could possibly be reflected in the transmitter release evoked by
depolarization. We therefore studied the effects of GABA release evoked
by KCl-induced depolarization of the synaptic boutons attached to the
medial preoptic neurons. When the cells with synaptic boutons are
transiently perfused with 140 mM KCl, postsynaptic GABA-mediated
currents can be recorded under voltage-clamp conditions. At a steady
voltage, in a range of 34 mV to +6 mV, these bicuculline-sensitive
currents show a major transient outward component that is most likely
due to the synchronized exocytosis of GABA from several release sites
(Haage et al. 1998
). In control experiments without
steroid application, the current evoked by KCl usually reached a peak
within 30-130 ms and showed a subsequent roughly exponential decay
with a time constant of 60 ± 7 ms (n = 8). With
allopregnanolone present >30 s before the KCl application, the time
course of decay was prolonged [Fig. 6;
time constant 362 ± 85 ms (n = 5) in 2.0 µM
allopregnanolone; 195 ± 9 ms (n = 3) in 200 nM
allopregnanolone]. The amplitude of the KCl-evoked current was not
significantly affected, neither by 200 nM nor by 2.0 µM
allopregnanolone. The allopregnanolone effect was thus qualitatively
similar to the effect on spontaneous mIPSCs, described above.
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DISCUSSION |
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In the present work, the neurosteroid allopregnanolone was shown
to dramatically affect the currents evoked by GABA in acutely dissociated neurons of the medial preoptic nucleus. Although these cells are believed to be an important target for steroid actions (see,
e.g., Herbison 1997), effects of steroids on membrane
currents in preoptic neurons have, to our knowledge, previously not
been investigated.
Effect of allopregnanolone depends on GABA concentration
Allopregnanolone was here shown to reduce the currents evoked by
100 µM GABA, but to potentiate those evoked by 1.0 µM GABA. The
dependence on GABA concentration suggests that the strong potentiation
reported in earlier studies may partly be due to the use of a
relatively low GABA concentration. However, at present we cannot
exclude that differences in GABAA receptor
subunit composition partly contributes to the differences in effect of
allopregnanolone among the studies reported, since -,
-, and
-subunits have all been reported to influence the effect of
allopregnanolone (Belelli et al. 1996
; Brussaard
et al. 1997
; Davies et al. 1997
; Hauser
et al. 1996
; Lan et al. 1991
; Shingai et
al. 1991
; see also Smith et al. 1998
).
In the present study, allopregnanolone at a concentration of 2.0 µM
also induced membrane currents in the complete absence of externally
applied GABA. The shift in reversal potential with Cl concentration implies that these currents
can be attributed to Cl
ions, and the effect of
bicuculline further suggests that they were due to
GABAA receptor activation. This is consistent
with several earlier studies reporting that allopregnanolone in
micromolar concentration may directly activate the
GABAA receptor (Majewska et al.
1986
; Puia et al. 1990
; Shingai et al.
1991
).
Desensitization as mediator of steroid effects?
Not only the peak current amplitude, but also the current kinetics
was affected by allopregnanolone. The prolongation of the deactivation,
off, was the most prominent effect seen when
100 µM as well as when 1.0 µM GABA was used. It has been shown that transitions to and from a desensitized state may strongly affect the
time course of GABAA receptor deactivation
(Jones and Westbrook 1995
). In accordance with this
idea, it was recently suggested that another neurosteroid,
3
,21dihydroxy-5
-pregnan-20-one, prolonged GABAA receptor deactivation in cerebellar granule
cells mainly by reducing the rate of recovery from desensitization
(Zhu and Vicini 1997
).
Interestingly, in the latter study the steroid also reduced the peak
current evoked by (1 mM) GABA. Also this effect was suggested as due to
desensitization, of part of the available receptor population as a
consequence of preapplication of the steroid (Zhu and Vicini 1997). However, although an increased rate of desensitization was reported in the present study, a similar mechanism could not account for all our results. Thus desensitization of part of the available receptor population would not be expected to cause the increased rate of activation and increased peak current at 1.0 µM
GABA as reported here. Neither can effects on desensitization explain
the currents evoked by allopregnanolone in the absence of GABA.
Further, at the single-channel level, an increased desensitization should be seen as an increased duration of closed intervals or as a
reduced duration of open intervals in the presence of agonist, which is
in disagreement with reported effects of steroids (Twyman and
Macdonald 1992
; Zhu and Vicini 1997
). It is thus
clear that different or more complex models are required to account for
even the most obvious effects of steroid interaction with the
GABAA receptor.
It should further be clear that not only transitions to and from
desensitized states are of importance in shaping the "macroscopic" deactivation. Thus, for instance, alterations in rate constants involved in ligand binding alone affect deactivation as well as activation and desensitization in a detailed model of
GABAA receptor function (Gingrich et al.
1995). There should thus be room for alternative models of
neurosteroid interaction with GABAA receptors.
Effects of allopregnanolone on synaptic GABA-mediated currents
The results reported above imply that the effect of
allopregnanolone on GABA-mediated transmission in the CNS do not only depend on the cell type and receptor composition studied, but also on
the concentration of GABA in the synapse. Thus, if the GABA
concentration varies in different synapses, as has been suggested for
synapses of different cleft volume (Nusser et al. 1997),
it seems likely that the allopregnanolone effect will vary even if the
GABA receptors are identical. Further, because, as shown above, the
current kinetics is dramatically affected by allopregnanolone, differences in time course of GABA concentration at the receptors may
be important. It should thus be clear that it is essential, for
understanding the modulation caused by allopregnanolone, to study the
effects on synaptically released GABA, that is, in a physiological
concentration and time course within the synapse.
In contrast to the majority of previous studies of allopregnanolone
effects, carried out on cultured cells or on heterologous expression
systems, we used an acute preparation with functional synapses and thus
had the possibility to study synaptic currents (mIPSCs). The results
presented above showed that, in GABAergic synapses on MPN neurons,
allopregnanolone did not significantly affect the mIPSC amplitude, but
the decay time course was dramatically prolonged. A lack of
potentiation of mIPSC amplitude is consistent with the current
reduction reported here for exogenous application of 100 µM GABA and
recent reports that GABA may reach near millimolar concentrations
during synaptic transmission (see Edwards 1995, for
review). The prolongation of synaptic currents imply that allopregnanolone will affect the temporal characteristics of synaptic integration. Thus, although a single inhibitory synaptic current will
not be larger in amplitude, the prolonged duration implies that
temporal summation of inhibitory signals will be favored.
The above analysis of the allopregnanolone-induced prolongation of
mIPSC decay demonstrated that allopregnanolone is effective already in
concentrations as low as 6.3-20 nM (half-maximal effect at ~50 nM).
It has earlier been demonstrated that allopregnanolone concentrations
as high as 20 nM may be rapidly (<5 min) reached in the male rat brain
after exposure to ambient temperature swim stress (Purdy et al.
1991). It thus seems likely that allopregnanolone effects on
neurotransmission similar to those demonstrated in the present work may
occur under physiological conditions.
Presynaptic steroid effects
In the present study, allopregnanolone affected not only the time
course of mIPSCs but also the frequency of mIPSCs. The mIPSC amplitude
distribution, however, was not affected (Fig. 3), implying that the
observed increase in frequency was of similar magnitude for mIPSCs of
all amplitudes. Thus the effect on mIPSC frequency could not be
explained as merely apparent due to increased mIPSC amplitudes, but was
interpreted as an effect on the frequency of GABA release due to a
presynaptic steroid action. Although the effect was not prominent in
all cells, it was dramatic in some cases (Fig. 4A).
Previously, not much attention has been paid to the possible
presynaptic effects of allopregnanolone. However, recently
Poisbeau et al. (1997) reported an
allopregnanolone-induced increase in mIPSC frequency in cultured
pituitary cells, and a similar effect of pregnanolone in
Xenopus embryos was reported by Reith and Sillar
(1997)
. Thus our findings suggest that allopregnanolone may
also modulate transmitter release from terminals on MPN neurons in an
acute mammalian preparation.
Although our results imply an increased probability of spontaneous transmitter release, we found no significant effect of allopregnanolone on the amplitude of KCl-evoked synaptic current. Thus there is no evidence for steroid-mediated potentiation of the probability of transmitter release that is triggered by depolarization. The functional consequence of the presynaptic steroid action is an increase in "basal" (spontaneous) inhibition that adds to the postsynaptic steroid affect discussed above.
Conclusions
In conclusion, the results presented above demonstrate that the neurosteroid allopregnanolone modulates GABAA receptor-mediated currents in medial preoptic neurons in a manner that depends critically on GABA concentration and involves kinetic changes of activation/deactivation as well as of desensitization. In functional GABAergic synapses on MPN neurons, allopregnanolone modulates transmission via presynaptic as well as postsynaptic mechanisms. The modulatory effect is not a simple amplification of the synaptic signal, but implies altered temporal characteristics in terms of a prolonged time course of postsynaptic currents and an increased frequency of spontaneous synaptic signals. A full understanding of the precise role of allopregnanolone-induced synaptic modulation for the main functions ascribed to preoptic neurons, regulation of sexual behavior, thermoregulation, and slow-wave sleep, will require further investigation.
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ACKNOWLEDGMENTS |
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This work was supported by Swedish Medical Research Council Project No. 11202, Gunvor och Josef Anérs Stiftelse, Magn. Bergvalls Stiftelse, Åke Wibergs Stiftelse, Umeå University, and a "Spjutspets" grant from Umeå Sjukvård.
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FOOTNOTES |
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Address reprint requests to S. Johansson.
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 14 December 1998; accepted in final form 15 March 1999.
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REFERENCES |
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