1Department of Psychology, Loyola Marymount University, Los Angeles, California 90045; 2Program in Neuroscience, University of Southern California, Los Angeles, California 90089; and 3Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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Foy, M. R.,
J. Xu,
X. Xie,
R. D. Brinton,
R. F. Thompson, and
T. W. Berger.
17-estradiol enhances NMDA receptor-mediated EPSPs and long-term
potentiation. Gonadal steroid hormones influence CNS
functioning through a variety of different mechanisms. To test the
hypothesis that estrogen modulates synaptic plasticity in the
hippocampus, in vitro hippocampal slices from 2-mo-old Sprague-Dawley
male rats were used to determine the effect of 17
-estradiol on both N-methyl-D-aspartate (NMDA) receptor-mediated
excitatory postsynaptic potentials (EPSPs) through intracellular
recordings and long-term potentiation (LTP) through extracellular
recordings. Intracellular EPSPs and extracellular field EPSPs (fEPSPs)
were recorded from CA1 pyramidal cells by stimulating Schaffer
collateral fibers. In intracellular experiments, slices were perfused
with medium containing bicuculline (5 µM) and low Mg2+
(0.1 mM) to enhance the NMDA receptor-mediated currents and
6,7-dinitroquinoxaline-2,3-dione (DNQX) (10 µM) to block the
-amino-3-hydroxy-5-methyl-4-isoxazoleproprianate (AMPA)
receptor-mediated component. The effects of 17
-estradiol on NMDA
receptor-mediated activity were excitatory; concentrations >10 nM
induced seizure activity, and lower concentrations (1 nM) markedly
increased the amplitude of NMDA-mediated EPSPs (both the first and
second responses increased during paired pulse stimulation by 180 and
197%, respectively). In extracellular experiments, slices perfused
with 17
-estradiol (100 pM) exhibited a pronounced, persisting, and
significant enhancement of LTP of both the fEPSP slope (192%) and
fEPSP amplitude (177%) compared with control slices (fEPSP slope = 155%; fEPSP amplitude = 156%) 30 min after high-frequency
stimulation. These data demonstrate that estrogen enhances NMDA
receptor-mediated currents and promotes an enhancement of LTP
magnitude.
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INTRODUCTION |
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Estrogenic steroids are reported to alter
electrophysiological, biochemical, and morphological properties of
mammalian CNS neurons and glial cells (Brinton 1993;
Brinton et al. 1997a
,b
; Gould et al.
1990
; Murphy and Segal 1996
; Simerly et
al. 1990
; Stone et al. 1998
; Wong and
Moss 1991
; Woolley et al. 1990
). Although modification of gene expression as a consequence of estrogen liganding to DNA-binding receptors is the traditional framework for interpreting underlying mechanisms, an increasing number of reports document effects
of acute application of estrogenic steroids that are too rapid
(occurring within
10 min) to be accounted for by a genomic pathway.
In particular, estrogenic steroid-induced changes in neuronal
excitability suggest other, nongenomic mechanisms involving a direct
interaction with sites of the plasma membrane to regulate ligand-gated
ion channels and neurotransmitter transporters (Wong et al.
1996
). Effects of estrogen on the electrophysiological activity
of rodent hippocampal neurons were first reported by Teyler et al., who
found that 17
-estradiol treatment induced a rapid (<10 min)
enhancement of glutamatergic synaptic transmission in the CA1 region of
in vitro hippocampal slices (Teyler et al. 1980
).
Subsequent reports indicated that only the biologically active isomer
of estrogen, 17
-estradiol, and not the 17
-estradiol isomer is
effective in eliciting these short-term electrophysiological effects
(Foy and Teyler 1983
; Wong and Moss 1991
,
1992
). On the basis of intracellular in vitro recordings
from CA1 pyramidal cells, Wong and Moss (1992)
reported that
administration of 17
-estradiol increased synaptic excitability by
enhancing the magnitude of
-amino-3-hydroxy-5-methyl-4-isoxazoleproprianate (AMPA)
receptor-mediated responses. The rapid onset of increased excitability
and its blockade by 6-cyano-7-nitroquinaxaline-2,3-dione (CNQX, an AMPA
receptor antagonist) and not D-2-amino-5-phosphonovalerate
[D-APV, a competitive N-methyl-D-aspartate (NMDA) receptor
antagonist] supported a postsynaptic membrane site of action and an
expression by non-NMDA receptor-channels. Later studies with whole cell
recordings found that acute 17
-estradiol application potentiated
kainate-induced currents in a subpopulation (38%) of CA1 cells
(Gu and Moss 1996
), although a direct interaction between 17
-estradiol and the receptor channel was not indicated (Wong and Moss 1994
). In total, the work of Moss and
colleagues supports the possibility of a second messenger mechanism
underlying the rapid effects of 17
-estradiol, most likely involving
a G-protein coupled AMPA-dependent phosphorylation event.
The apparently exclusive estrogenic steroid modification of non-NMDA
receptor channels stands in contrast to a large body of evidence
demonstrating 17-estradiol regulation of NMDA receptor-mediated function. Morphological studies on the course of neuronal development conducted in vitro in our laboratory (Brinton et al.
1997a
,b
) have shown that estrogenic steroids exert a
growth-promoting, neurotrophic effect on hippocampal and cortical
neurons via a mechanism that requires activation of NMDA receptors.
Moreover, the neurotrophic effects of estrogenic steroids can be
blocked by an NMDA receptor antagonist in cultured neurons before
synaptic contacts occur (Brinton et al. 1997b
). In vivo
studies by Woolley and McEwen (1994)
revealed a proliferation of
dendritic spines after 17
-estradiol treatment that can be prevented
by blockade of NMDA receptor channels, although not by AMPA or
muscarinic receptor antagonists. Other reports provide evidence that
chronic 17
-estradiol treatment increases the number of NMDA receptor binding sites and NMDA receptor-mediated responses (Gazzaley et al. 1996
; Woolley et al. 1997
).
The possibility of direct regulation of NMDA receptor-mediated synaptic
transmission by 17-estradiol may not have been detected previously
because tests of this hypothesis are so few in number and, more
importantly, have yet to be conducted under optimal conditions. Because
of the voltage-dependent blockade of the NMDA channel by
Mg2+ and the slow kinetics of ligand-gated channel opening
relative to that of the AMPA receptor subtype, there is only a minor
NMDA receptor-mediated component of the excitatory postsynaptic
potential (EPSP) evoked by low-frequency stimulation of glutamatergic
afferents. This NMDA component can be enhanced with low
Mg2+ concentration or high-frequency stimulation to induce
the depolarization accompanying the summation of overlapping EPSPs
(Xie et al. 1992
). In the experiments reported here, we
used both the conditions of low Mg2+ and high-frequency
stimulation in separate experiments to examine the acute effects of
17
-estradiol on pharmacologically isolated NMDA receptor-medicated
EPSPs in CA1 pyramidal cells to determine if estradiol alters NMDA
receptor-channel activity. In other experiments, we investigated the
acute effects of 17
-estradiol treatment on the induction and
expression of long-term potentiation (LTP), an enduring enhancement of
glutamatergic synaptic transmission that, in the hippocampal CA1
region, requires high-frequency stimulation sufficient to activate NMDA
receptor-channels.
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METHODS |
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Transverse hippocampal slices (400 µm) were prepared from
experimentally naive, 200- to 350-g adult male Sprague-Dawley rats (Harlan). Slices were incubated in an artificial cerebrospinal fluid
(aCSF) perfusion medium that consisted of (in mM) 126 NaCl, 5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 2 CaCl2, 2 MgCl2, 2 ascorbic acid, bubbled with
95% O2-5% CO2 and maintained at 32 ± 0.5°C. For intracellular recordings, slices were superfused (3
ml/min) in a submerged chamber with aCSF containing bicuculline (5 µM) and either the AMPA receptor antagonist
6,7-dinitroquinoxaline-2,3-dione (DNQX) (10 µM) and a reduced
Mg2+ concentration (0.1 mM) to isolate NMDA
receptor-mediated EPSPs or the NMDA receptor antagonist
D-2-amino-5-phosphonovalerate (D-APV; 50 µM)
and a higher Mg2+ concentration (1 mM) to isolate AMPA
receptor-mediated EPSPs. CA1 pyramidal cell EPSPs were recorded
intracellularly with glass microelectrodes filled with 2 M potassium
acetate (resistance: 90-150 M
). Depolarizing and hyperpolarizing
current injections were applied for measurement of input resistance to
generate voltage steps across the neuronal membrane for calculation of
input resistance. Experiments were conducted only when stable
intracellular recordings were obtained from neurons with a resting
membrane potential of at least
55 mV, overshooting action potentials,
and a minimum input resistance of 20 M
(Schwartzkroin
1975
). Throughout the experiment, EPSPs were evoked via
Schaffer collateral stimulation and recorded at 0.1 Hz, with
17
-estradiol added to the aCSF incubating the slices after a stable,
10-min baseline period. In Figs.
1 and 2,
each response shown is an average of 10 consecutive, individual EPSPs,
except the single, individual action potential responses. For
extracellular recordings, slices were maintained in an interface chamber continuously perfused (
2 ml/min) for
1 h with aCSF
containing 1 mM Mg2+. Field EPSPs (fEPSPs) were recorded
from stratum radiatum of CA1 with glass microelectrodes filled with 2 M
NaCl. Schaffer collateral/commissural axons were stimulated (0.05 Hz)
at intensities adjusted to produce an evoked response that was 50% of
the maximum recorded fEPSP for each recording with a bipolar nichrome
electrode placed in the proximal stratum radiatum. After 10 min of
stable, baseline fEPSP recordings, slices continued to be perfused with aCSF or aCSF containing 17
-estradiol (Sigma Chemical) in
concentrations indicated in RESULTS. The effect of
17
-estradiol on LTP was studied with high-frequency stimulation
(after 30 min of 17
-estradiol perfusion), consisting of five trains
of 100-Hz stimulation, with each train having a duration of 200 ms; the
intertrain interval was 10 s. During the post-high-frequency
stimulation period, fEPSPs were recorded from control slices (aCSF
perfusion) and experimental slices (aCSF + 17
-estradiol perfusion)
for 30 min.
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Data for both intracellular and extracellular recordings were collected and analyzed on a PC with programs written in AXOBASIC. The initial slope and amplitude of EPSPs were used in all statistical analyses, with data from different slices combined and normalized to compare recordings from the control and experimental periods. Statistical significance of differences in normalized (means ± SE) EPSP slope and amplitudes within the previous conditions were evaluated with analyses of variance and planned two-tailed t-tests.
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RESULTS |
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17-estradiol enhancement of NMDA receptor-mediated EPSPs
In the presence of the AMPA receptor antagonist DNQX and 0.1 mM
Mg2+, EPSPs evoked by Schaffer collateral stimulation were
prolonged in duration, with slow rise and fall times characteristic of
NMDA receptor-mediated synaptic responses (Xie et al.
1992). Identification of DNQX-resistant responses as being NMDA
receptor mediated was confirmed by the effects of D-APV,
which completely abolished residual evoked synaptic activity (Fig. 1).
At 1 nM, 17-estradiol induced a rapid increase in the amplitude of
the NMDA receptor-mediated EPSPs evoked by paired impulse stimulation
of Schaffer collateral input in 9 of 13 cells. Measured at ~6 min
after application of 17
-estradiol, the enhancement occurred for both
of the paired responses (Fig. 1C). The mean increase of the
first response was 180 ± 26%, P < 0.05, and
that of the second response was 197 ± 22%, P < 0.01. After correcting for the lag time of the perfusion system (~1.5
min), the latency for onset of the 17
-estradiol effect was <2 min
for all cells. In some cells, the enhanced EPSPs reached the firing
threshold after a longer (>10 min) 17
-estradiol application period
(Fig. 1B). For two cells, the enhanced EPSPs were further
confirmed as NMDA receptor mediated by their nearly complete blockade
after bath application of 50 µM D-APV (Fig.
1B).
In another series of experiments, the AMPA receptor was
pharmacologically isolated by applying the NMDA receptor antagonist D-APV (50 µM) to the bath. The effect of 17-estradiol
on the AMPA receptor was then examined, and we observed potentiation of
the AMPA component by 17
-estradiol in 5 of 14 cells (36%) (Fig. 2,
A and B). This result is consistent with a
previous report (Wong and Moss 1991
).
17-estradiol enhancement of LTP
Consistent with the results of intracellular studies, an increase
in synaptic transmission occurred in CA1, after acute perfusion of 100 pM 17-estradiol onto experimental hippocampal slices (fEPSP mean
increase of slope was 113% (experimental) vs. 101% (control); fEPSP
mean increase of amplitude was 112% (experimental) vs. 100% (control), F(1,180) = 229, P < 0.0001, and F(1,180) = 353, P < 0.0001, respectively). Field EPSP responses of the two groups were identical
during the baseline period before 17
-estradiol perfusion [fEPSP
slope and fEPSP amplitude, F(1,58) = 0.000, NS, and
F(1,58) = 0.000, NS]. In Fig.
3, the two groups of slices from normal,
young adult male rats (17
-estradiol-treated vs. aCSF-control) showed
no significant differences in either their measured fEPSP slopes or
fEPSP amplitudes over the 10-min baseline period. The increase in
synaptic transmission began to develop ~3-4 min after
17
-estradiol perfusion onto the experimental slices and continued
throughout the 30-min perfusion period.
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When LTP was assessed after high-frequency stimulation, fEPSP
slopes and amplitudes were increased significantly for the
17-estradiol-treated slices compared with control slices. fEPSP
mean increase of slope was 192% (experimental) vs. 154% (control);
fEPSP mean increase of amplitude was 176% (experimental) vs. 156%
(control), F(1,200) 305.86, P < 0.0001, and
F(1,200) 113.58, P < 0.0001, respectively. Thus hippocampal slices treated with 17
-estradiol exhibited a pronounced, persisting, and significant increase in LTP as measured by
both population fEPSP slope and fEPSP amplitude recordings.
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DISCUSSION |
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These experiments establish several fundamental characteristics of
the effects of estrogen on synaptic transmission in the mammalian CNS.
First, we demonstrate that estrogen acts rapidly via presumed membrane
mechanisms to enhance both NMDA and AMPA receptor/channel processes in
response to glutamate released from Schaffer collateral terminals. Wong
and Moss (1992) reported that estradiol enhances the amplitude of EPSPs
at the Schaffer collateral-CA1 synapse. They observed that the increase
in EPSP amplitude remains unchanged in the presence of
D-APV but was blocked by CNQX, suggesting the NMDA receptor
irrelevant in regard to the effects of estradiol on synaptic
transmission. However, even for AMPA receptor-mediated EPSPs, the
enhancing effect of estradiol was only seen in 36% of their cells.
Furthermore, the NMDA component accounts for only a small fraction of
the total EPSP profile under their experimental conditions.
Consequently, in such conditions, any effect of estradiol on NMDA
receptor/channel function would be minimally expressed and may be
undetectable. In fact, in our experiments under conditions in which a
lowered extracellular concentration of Mg2+ was applied,
estradiol led to a higher percentage enhancement of the NMDA component
compared with the AMPA component (75 vs. 36%). The experimental
conditions used here, i.e., pharmacological isolation of the two types
of receptors, also ruled out the possibility of the enhancement of one
receptor component as the result of the other. Thus estradiol seems to
act on both NMDA and AMPA receptors and produce acute effects in a
similar fashion. It seems unlikely that these effects are presynaptic;
estrogen can induce an increase in the number of stimulus-evoked action
potentials in the Schaffer collaterals or an increase in the amount of
glutamate released per Schaffer collateral action potential, but this
possibility was not definitely ruled out here or in earlier studies. It
is not yet known whether these estrogen effects are due to an action directly on the receptors or indirectly via second messenger processes that in turn influence NMDA and AMPA receptor/channel processes.
Second, our results indicate that estradiol can both increase synaptic
transmission in the hippocampus and markedly enhance LTP in CA1 neurons
of adult, male rats. The enhancement of LTP after acute 17-estradiol
application (Fig. 3) could be due to an increase in activation of NMDA
receptors/channels or an increase in AMPA receptor function. Both
possibilities are consistent with our intracellular data. Whatever the
mechanism, the fact is that estrogen enhances LTP in hippocampal CA1.
To the extent that LTP is a mechanism involved in processes of coding
and storage of information, i.e., in memory formation, estrogen
enhances these processes. Indeed, the estrogen enhancement of LTP
reported here suggests a possible mechanism whereby estrogen can exert
its facilitatory effects on memory processes in humans. Recent clinical
evidence indicates that estrogenic steroids can enhance cognitive
functions in humans, particularly in postmenopausal women
(Henderson et al. 1997
; Kawas et al.
1997
). Although the estrogen regulation of functions within the
limbic system may result mostly from the classical genomic mechanism,
an acute, nongenomic effect of estrogen could provide an additional
short-term mechanism in modulating synaptic transmission and
plasticity. Our studies demonstrate that estradiol enhances synaptic
transmission through both NMDA and AMPA receptors/channels and that
these enhancements may underlie its facilitatory effect on the
magnitude of LTP.
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ACKNOWLEDGMENTS |
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We are grateful for critical comments and advice from M. Baudry and C. Finch of the University of Southern California, and J. Foy and K. Krekorian of Loyola Marymount University.
This work was supported by National Institutes of Health Grants AG-05142, AG-14751, MH-00343, and MH-51722, by National Science Foundation Grant IBN 9215069, and by Human Frontiers Organization, Loyola Marymount University, Sankyo Corporation, and Alzheimer's Association.
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FOOTNOTES |
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Address for reprint requests: M. R. Foy, Dept. of Psychology, Loyola Marymount University, 7900 Loyola Blvd., Los Angeles, CA 90045.
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 11 September 1998; accepted in final form 30 October 1998.
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REFERENCES |
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