1Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892; and 2Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Tokyo 108, Japan
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ABSTRACT |
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Pozzo-Miller, Lucas D.,
Takafumi Inoue, and
Diane Dieuliis Murphy.
Estradiol increases spine density and NMDA-dependent Ca2+
transients in spines of CA1 pyramidal neurons from hippocampal slices. To investigate the physiological consequences of the increase in spine density induced by estradiol in pyramidal neurons of the
hippocampus, we performed simultaneous whole cell recordings and
Ca2+ imaging in CA1 neuron spines and dendrites in
hippocampal slices. Four- to eight-days in vitro slice cultures were
exposed to 17-estradiol (EST) for an additional 4- to 8-day period,
and spine density was assessed by confocal microscopy of DiI-labeled
CA1 pyramidal neurons. Spine density was doubled in both apical and
basal dendrites of the CA1 region in EST-treated slices; consistently,
a reduction in cell input resistance was observed in EST-treated CA1
neurons. Double immunofluorescence staining of presynaptic
(synaptophysin) and postsynaptic (
-subunit of CaMKII) proteins
showed an increase in synaptic density after EST treatment. The slopes
of the input/output curves of both
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and
N-methyl-D-aspartate (NMDA) postsynaptic
currents were steeper in EST-treated CA1 neurons, consistent with the
observed increase in synapse density. To characterize NMDA-dependent
synaptic currents and dendritic Ca2+ transients during
Schaffer collaterals stimulation, neurons were maintained at +40 mV in
the presence of nimodipine, picrotoxin, and
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). No differences in resting
spine or dendritic Ca2+ levels were observed between
control and EST-treated CA1 neurons. Intracellular Ca2+
transients during afferent stimulation exhibited a faster slope and
reached higher levels in spines than in adjacent dendrites. Peak
Ca2+ levels were larger in both spines and dendrites of
EST-treated CA1 neurons. Ca2+ gradients between spine heads
and dendrites during afferent stimulation were also larger in
EST-treated neurons. Both spine and dendritic Ca2+
transients during afferent stimulation were reversibly blocked by
D,L-2-amino-5-phosphonovaleric acid (D,L-APV).
The increase in spine density and the enhanced NMDA-dependent
Ca2+ signals in spines and dendrites induced by EST may
underlie a threshold reduction for induction of NMDA-dependent synaptic
plasticity in the hippocampus.
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INTRODUCTION |
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The function of dendritic spines has been studied
using various structural, biophysical, and computational approaches
(Shepherd 1996). Dendritic spines may act as independent
compartments with a diffusional barrier that allows for biochemical
gradients to be generated between spines and dendrites (Koch and
Zador 1993
; Svoboda et al. 1996
; Wickens
1988
). In this model, voltage-gated ionic influx and/or
signaling molecules generated by synaptic activation of membrane
receptors coupled to second-messenger cascades would be restricted to
activated spines and therefore used for integrative computation of
synaptic inputs (Llinás 1995
). Synaptically mediated, compartmentalized intracellular Ca2+ transients
in CA1 hippocampal dendritic spines (Alford et al. 1993
;
Müller and Connor 1991
; Petrozzino et al.
1995
; Yuste and Denk 1995
) have been postulated
to participate in the induction of
N-methyl-D-aspartate (NMDA)-dependent long-term
synaptic modifications, such as long-term potentiation (LTP)
(Bliss and Collingridge 1993
).
Dendritic spine density in hippocampal CA1 pyramidal neurons increases
during the estrous cycle coincident with the estrogen peak
(Woolley et al. 1990), and 17
-estradiol (EST)
augments hippocampal spine density in vivo (Woolley and McEwen
1994
) and in vitro (Murphy and Segal 1996
).
Here, we show that CA1 pyramidal neurons from postnatal hippocampal
slices retain the ability to increase their dendritic spine density in
response to estradiol treatment in vitro, as observed in embryonic
hippocampal neurons in dissociated cultures (Murphy and Segal
1996
). Because LTP is enhanced as estradiol levels are higher
during the estrous cycle (Warren et al. 1995
), and EST
facilitates the induction of LTP in ovariectomized (OVX) awake rats
(Córdoba Montoya and Carrer 1997
), increases in
dendritic spine density may underlie enhanced hippocampal synaptic
plasticity. To investigate the physiological consequences of the
EST-induced increase in spine density, we performed simultaneous whole
cell recordings and digital imaging of NMDA-dependent intracellular Ca2+ transients in spines and dendrites of CA1 pyramidal
neurons in hippocampal slices during high-frequency Schaffer
collaterals stimulation.
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METHODS |
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Slice cultures
Hippocampal slices (500 µm thick) from postnatal day
7 rats were prepared with a custom-designed wire slicer
(Katz 1987) and maintained in vitro in a 36°C, 5%
CO2 humidified incubator, as previously described
(Petrozzino et al. 1995
; Pozzo-Miller et al.
1993
, 1995
, 1996
). All tissue
culture reagents were obtained from GIBCO BRL (Gaithersburg, MD). Four-
to eight-days in vitro (div) organotypic slice cultures were exposed to
EST (0.2 µg/ml; Sigma, St. Louis, MO) for an additional 4- to 8-day
period. Control and EST-containing media were replaced every 48 h.
Dendritic spine labeling, confocal imaging, and quantification
Eight- to 16-div control and EST-treated slices were fixed
overnight in 4% paraformaldehyde in PBS. After rinsing in PBS, small
droplets (<10 µm) of a saturated solution of
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI; Molecular
Probes, Eugene, OR) in fish oil were microinjected with a patch pipette
into the CA1 region using a picospritzer (PLI-100; Medical Systems,
Greenvale, NY). After 12-24 h at 4°C in the dark,
z-sections from labeled dendritic segments were collected in
a laser-scanning confocal microscope (Zeiss, Thornwood, NY; ×100, 1.4 NA oil immersion objective). Dendritic projections shorter than 1-3
µm were quantified off-line as dendritic spines (Murphy and
Segal 1996). Due to the high density of labeled dendrites, the
single sections from the z-section projections were used to count the number of spines for measured lengths of dendrite (10-µm segments), ensuring that each spine was counted only once by following its course in the z-section reconstructions. Spine counts
were performed in a total of 164 dendritic segments (72 apical and 92 basal) from control slices, and in 112 segments from EST-treated slices
(98 apical dendrites and 14 basal dendrites).
Double immunofluorescent staining and confocal imaging
Eight to 16-div control and EST-treated slices were fixed
overnight in 4% paraformaldehyde in PBS. After incubation in blocking and permeabilization buffer containing 10% serum in PBS with 0.1% saponin, slices were exposed to primary antibodies against
synaptophysin (Zymed, South San Francisco, CA) and the -subunit of
Ca2+/calmodulin-dependent protein kinase 2 (CamKII,
Boeringer-Mannheim, Indianapolis, IN) for 1 h at room temperature.
After incubation in FITC- and rhodamine-conjugated secondary antibodies
for 1 h at room temperature, slices were finally mounted and
sealed using Vectashield (Vector Laboratories, Burlingame, CA). Imaging
was performed in a confocal laser scanning microscope (Zeiss, ×100, NA
1.4 oil immersion objective).
Simultaneous whole cell recording and Ca2+ imaging
Eight- to 16-div control and EST-treated slice cultures were continuously perfused (2 ml/min) in an immersion-type slice chamber with artificial cerebrospinal fluid (ACSF) at room temperature containing (in mM) 124 NaCl, 2 KCl, 1.3 MgSO4, 1.24 KH2PO4, 17.6 NaHCO3, 2.5 CaCl2, and 10 D-glucose; 310-320 mOsm; equilibrated with 95% O2-5% CO2. Nimodipine (20 µM in 0.05% DMSO; RBI, Natick, MA) and picrotoxin (50 µM; Sigma) were routinely included in the ACSF, while D,L-2-amino-5-phosphonovaleric acid (D,L-APV; 100 µM; Sigma) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 µM in 0.1% DMSO; RBI) were added when indicated.
CA1 pyramidal neurons in the ~200-µm-thick slice cultures were
visualized by differential interference contrast (DIC) optics with
infrared illumination (centered at 770 nm, 40 nm bandwidth; Omega
Optical, Brattleboro, VT) in a fixed-stage upright microscope (Zeiss
Axioskop FS) using a water-immersion ×63 objective (Zeiss Achroplan,
0.9 NA), a charge-coupled device (CCD) camera (C2400; Hamamatsu,
Hamamatsu City, Japan), and video monitor (Dodt and Zieglgänsberger 1990). Somatic whole cell recordings were
performed as described (Petrozzino et al. 1995
;
Pozzo-Miller et al. 1993
, 1995
,
1996
) using unpolished patch pipettes pulled from
thin-wall borosilicate glass capillary (WPI, Sarasota, FL), and
containing (in mM) 120 Cs+-gluconate, 10 NaHEPES, 17.5 CsCl, 10 NaCl, 2 Mg-ATP, 0.2 Na-GTP, 5 QX-314, 0.5 hexapotassium salt
of the Ca2+-sensitive dye bis-fura-2
(Kd = 525 nM; Molecular Probes); 280-290 mOsm;
pH 7.2 (final resistance 4-8 M
). Neurons were voltage clamped in
the continuous, single-electrode mode of an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA). Excitatory postsynaptic currents (EPSCs) were elicited with a bipolar stainless steel electrode (WPI),
or a patch pipette filled with ACSF, positioned over the Schaffer
collaterals in CA1 stratum radiatum, 100-200 µm away from the
dye-filled neuron. Square current pulses of 100 µs were delivered by
a computer-triggered stimulus isolation/constant-current unit (Isolator
11; Axon Instruments). High-frequency afferent stimulation consisted of
1-s trains at 50 Hz. Voltage (Vh) and current
(I) signals were digitized at 10 kHz (ITC-16; Instrutech, Great Neck, NY) for display and analysis.
The fluorescent Ca2+-sensitive dye bis-fura-2 was excited
with 357- and 380-nm monochromatic light (12 nm bandwidth) using a galvanometric scanner-mounted grating (<1.5 ms wavelength change; Polychrome I; TILL Photonics, Planegg, Germany), under computer control
(PCI-1200; National Instruments, Austin, TX). Fluorescent dye emission
(dichroic beamsplitter mirror 420 nm, emission longpass filter
450
nm) was detected with a 12-bit, liquid-cooled, CCD camera (PXL-37;
Photometrics, Tucson, AZ) operating at
25°C in frame-transfer mode
(12-28 ms frame read-out interval for 100 × 200 pixel subarrays
with 3 × 1 pixel binning). Single frames obtained at the
calibrated isosbestic point (357 nm; Ca2+-insensitive
wavelength) were acquired before and after a fast sequence of 10-20
frames at 380 nm excitation (Ca2+-sensitive wavelength) to
generate ratio images. Synchronized afferent stimulation (1 s at 50 Hz)
was delivered after 2 prestimulus frames of the fast sequence at 380 excitation, and continued thereafter. Regions of interests (ROIs;
<4 × 6 pixels) were defined within spine heads and adjacent
dendrites. Only primary or secondary dendrites, 50-100 µm from the
soma, were selected to ensure that recordings were performed from
spines and adjacent dendritic segments that reached steady-state dye
concentration and appropriate signal-to-noise ratio in reasonably short
times (20-30 min) after whole cell break-in. This also allowed better
voltage clamp of synaptic currents originated from electrotonically
proximal regions. ROIs over the slice but outside the dye-filled neuron
were used for background estimation. Free cytosolic Ca2+
concentration was calculated from background-subtracted average fluorescence intensities within ROIs by the isosbestic ratio method (Grynkiewicz et al. 1985
; Neher and Augustine
1992
). Synchronized electrical and optical recording, afferent
stimulus trigger, and control of the cooled CCD camera trigger and the
galvanometric monochromator was performed using a single Power
Macintosh computer (9500/120 MHz, Apple Computer, Cupertino, CA)
running custom-developed software.
All data are expressed as means ± SE. Statistical differences were assessed by the Student's t-test. P < 0.05 was considered as significant.
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RESULTS |
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CA1 pyramidal neurons from postnatal hippocampal slices maintained in organotypic culture develop dendritic spines resembling those observed in vivo (Fig. 1, A and B). In DiI-labeled CA1 neurons from 8- to 16-div control slices (Fig. 1A), spine density was 11 ± 0.5 (SE) spines per 10 µm of dendritic length (n = 72 apical and 92 basal dendrites from 3 slices). In slices exposed for 4-8 days to 0.2 µg/ml EST (starting at 4-8 div; Fig. 1B), spine density was significantly larger, reaching 21 ± 0.4 spines per 10 µm of dendritic length (n = 98 apical and 14 basal dendrites from 5 slices; t-test, P < 0.0001; Fig. 2A). Because the same effect of EST on spine density was observed in apical and basal dendritic segments, all data were pooled for statistical analysis.
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Consistent with the increased spine density, and therefore membrane
surface area, cell input resistance (Rn) was
lower in EST-treated CA1 neurons. The contribution of spontaneous
synaptic activity to Rn was eliminated by
addition of picrotoxin, CNQX, and D,L-APV to block
GABAA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and NMDA receptors, respectively. GABAB
receptor activity was blocked by intracellular application of QX-314
and Cs+. Under voltage clamp at
80 mV in the whole cell
configuration with patch pipettes containing Cs+ and QX-314
to block K+ and Na+ channels, respectively,
control CA1 pyramidal neurons had input resistances of 368 ± 45 M
(n = 9 cells), whereas EST-treated neurons had
input resistances of 230 ± 15 M
(n = 13 cells;
t-test, P = 0.0035).
Do newly formed spines induced by EST in the postnatal hippocampal
slices establish synapses with functional presynaptic partners? The
density of presynaptic terminals assayed by immunostaining of
synaptophysin was indeed higher in EST-treated slices (Fig. 1,
C and D). Furthermore, synapse density was also
increased by EST, as evidenced by double immunofluorecent staining of
presynaptic (synaptophysin) and postsynaptic (-subunit of CaMKII)
proteins (Fig. 1, E and F). The ratio of pre- to
postsynaptic immunostaining (yellow represents the overlapping staining
in Fig. 1, E and F) was not different after EST
treatment, consistent with the hypothesis that new spines have
presynaptic partners. As suggested by these results, and other
structural studies (Murphy and Segal 1996
; Woolley and McEwen 1992
), newly formed dendritic spines
induced by estradiol may represent functional synaptic contacts. To
test this hypothesis, input/output (I/O) curves were constructed for the AMPA and NMDA components of the excitatory postsynaptic currents (EPSC) (Hestrin et al. 1990
). Figure 2B shows
AMPA-mediated EPSCs evoked at resting potentials (
80 mV) in the
presence of picrotoxin (50 µM), and NMDA-dependent EPSCs evoked at
depolarized potentials (+40 mV) after addition of CNQX (20 µM); the
L-type Ca2+ channel blocker nimodipine (20 µM) was also
included in the bath to reduce voltage-dependent Ca2+
influx evoked by the step depolarization to +40 mV. The I/O curves of
the AMPA EPSCs had a tendency to exhibit steeper slopes in EST-treated
slices than in control slices (2.27 n = 7 vs. 0.68 n = 3). Similarly, the I/O curve of the NMDA EPSCs
exhibited a trend of steeper slopes in EST-treated slices (2.67 n = 3 cells vs. 1.57 n = 4). Taken
together, these structural and physiological observations are
consistent with an increase of active synapses on spines containing
functional AMPA and NMDA receptors.
To investigate the effect of increased spine density on
NMDA-dependent spine and dendritic Ca2+ transients during
synaptic activity, simultaneous voltage clamp and Ca2+
imaging was performed in CA1 neurons using the Ca2+
indicator bis-fura-2. Only clearly resolvable, laterally projecting spines in primary or secondary dendrites, 50-100 µm from the soma, were included in the following analysis. To minimize voltage-gated Ca2+ influx into spines and dendrites during high-frequency
stimulation of Schaffer collaterals, CA1 neurons were dialyzed with
Cs+ and QX-314 and then steadily depolarized (soma voltage
+40 mV) in the presence of 20 µM nimodipine. At these potentials,
EPSCs are outward flowing, and negative voltage changes (toward the NMDA receptor reversal potential at 0 mV) would occur at poorly clamped
synaptic sites in spines and dendrites during high-frequency afferent
stimulation. Holding the cells at depolarized potentials would reduce
the likelihood of activation of voltage-gated Ca2+ channels
and, together with the blockade of AMPA (20 µM CNQX) and
GABAA (50 µM picrotoxin) receptors, allows for isolation
of NMDA-dependent Ca2+ transients (Malinow et al.
1994; Perkel et al. 1993
; Pozzo-Miller et
al. 1996
). After an initial widespread elevation when
Vh was stepped to +40 mV, Ca2+
concentration returned to levels that, although higher than those at
resting potential, were within the range allowing transients to be
measured without saturation of bis-fura-2. No differences in resting
Ca2+ levels of CA1 spines were observed between control
(282 ± 13 nM, n = 16, 7 cells) and EST-treated
neurons (296 ± 25 nM, n = 19, 8 cells;
t-test, P = 0.6543). Similar results were
obtained for CA1 dendrites (284 ± 11, n = 22, 7 control cells vs. 275 ± 12 nM, n = 27, 8 EST
cells; t-test, P = 0.595).
Under the above conditions, Schaffer collateral stimulation (1 s, 50 Hz) evoked outward EPSCs of similar amplitude (1.7 ± 0.5 nA
control cells vs. 1.3 ± 0.2 nA EST cells; t-test,
P = 0.4929). The associated spine and dendritic
Ca2+ transients were detected within 20-25 ms from the
stimulus onset and exhibited a faster slope in spine heads than in
adjacent dendritic segments (arrowheads in Fig.
3). Ca2+ transients reached
maximum levels at 100-200 ms from the stimulus onset in both
compartments (Fig. 3). In control CA1 cells (Fig. 3A),
maximum Ca2+ elevations at 100-200 ms from the stimulus
onset were larger in spine heads, reaching 933 ± 72 nM in spines
and 585 ± 51 nM in the adjacent dendrites (n = 16 spine/dendrite pairs from 7 cells; t-test, P = 0.0004). Similar Ca2+ spine/dendrite gradients were
observed in EST-treated CA1 cells (Fig. 3B), with peak
Ca2+ elevations reaching 1,436 ± 147 nM in spines and
972 ± 109 nM in the adjacent dendrites (n = 19 spine/dendrite pairs from 8 cells; t-test, P = 0.0156). Synaptic Ca2+ transients observed under these
conditions were reversibly blocked by 100 µM D,L-APV,
further confirming that they were mediated by activation of synaptic
NMDA receptors (Figs. 3 and 4).
D,L-APV also reversibly reduced the faster component of the
EPSCs, leaving a postsynaptic current previously characterized as a
nonselective cationic conductance mediated by G protein-coupled
metabotropic glutamate receptors (Pozzo-Miller et al.
1995).
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Maximum Ca2+ elevations in dendrites during afferent stimulation were larger in EST-treated CA1 cells (554 ± 39 nM, n = 22 dendrites from 7 control cells vs. 893 ± 87 nM, n = 27 dendrites from 8 EST cells; t-test, P = 0.0019; Fig. 2C). This observation is consistent with the prediction that a larger number of spines expressing functional Ca2+-permeable NMDA receptors permit larger Ca2+ influx into dendritic compartments. Surprisingly, peak Ca2+ elevations within spine heads were also larger in EST-treated CA1 cells (933 ± 72 nM, n = 16 spines from 7 control cells vs. 1,436 ± 147 nM, n = 19 spines from 8 EST cells; t-test, P = 0.0066; Fig. 2C). Due to this significant difference in peak spine Ca2+ elevations, spine/dendrite Ca2+ gradients had a tendency to be larger in EST-treated CA1 neurons (349 ± 40 nM, n = 16 spine/dendrite pairs from 7 control cells vs. 487 ± 83 nM, n = 19 spine/dendrite pairs from 8 EST cells; t-test, P = 0.1664).
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DISCUSSION |
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The current report presents direct structural and
physiological evidence that newly formed dendritic spines of CA1
pyramidal neurons induced by estradiol in postnatal hippocampal slices
represent functional postsynaptic compartments expressing AMPA and NMDA receptors. In addition to an increase in dendritic spine and synapse density, the sensitivity to AMPA and NMDA receptor-mediated synaptic input, and the NMDA-dependent Ca2+ transients in spines and
dendrites are enhanced in EST-treated CA1 pyramidal neurons. Our
results correlate well with recent observations that LTP is enhanced
during proestrous, when EST levels are highest (Warren et al.
1995), and that EST treatment facilitates the induction of LTP
in awake OVX rats (Córdoba Montoya and Carrer
1997
). These observations strongly suggest that increases in
dendritic spine density, together with enhanced NMDA-dependent spine
and dendritic Ca2+ signaling, may underlie augmented
hippocampal synaptic plasticity. Because the duration of the in vitro
EST treatment in the present report exceeds the EST peak during the
rodent estrous cycle by 3-7 days, our observations most likely reflect
organizational rather than activational EST effects during the adult
estrous cycle and, as such, may be relevant to synaptic development
and/or plasticity.
Compartmentalized NMDA-mediated Ca2+ transients in CA1
spines have been reported in earlier current-clamp experiments
(Müller and Connor 1991; Petrozzino et al.
1995
). Since then, several experimental approaches have been
used to minimize voltage-dependent Ca2+ influx and isolate
dendritic Ca2+ signals mediated by influx through NMDA
receptor channels during synaptic stimulation (Alford et al.
1993
; Malinow et al. 1994
; Perkel et al.
1993
; Pozzo-Miller et al. 1996
). In the present configuration the somatic membrane was steadily depolarized to +40 mV
under whole cell voltage clamp. If membrane voltage control were to
fail during afferent stimulation in the presence of nimodipine, CNQX,
and picrotoxin, the membrane potential would hyperpolarize toward 0 mV,
the reversal potential of the remaining glutamatergic currents. This
hyperpolarization would reduce the likelihood of activation of
voltage-activated Ca2+ channels. In addition, the
dihydropyridine Ca2+ channel antagonist nimodipine was
included in the ACSF cocktail to reduce the steady-state
Ca2+ influx through noninactivating Ca2+
channels, thus maintaining the background Ca2+ levels low.
Furthermore, Ca2+ imaging experiments were always performed
on primary dendrites, 50-100 µm from the cell body, ensuring
adequate voltage clamp of synaptic currents originated from
electrotonically proximal regions. Ca2+ transients still
occur in spines and adjacent dendrites under these stringent conditions
and, together with the associated EPSCs, were reversibly inhibited by
D,L-APV, confirming that they are mediated by the synaptic
activation of NMDA receptors. These observations have been recently
confirmed in layer V cortical pyramidal neurons using local photolysis
of caged glutamate to mimic presynaptic release and allowing complete
pharmacological blockade of voltage-dependent Ca2+ channels
(Schiller et al. 1998
).
The potential limitations of our experimental approach (unknown voltage
clamp of the spine membrane potential; altered activity of the
Na+/Ca2+ exchanger and prolonged
Ca2+ influx through NMDA receptors with altered kinetics at
depolarized potentials; contribution of Ca2+ release from
intracellular stores) do not affect the primary conclusion of our
imaging experiments: synaptic activation under conditions of minimal
voltage-gated Ca2+ influx and maximal NMDA receptor
activation is enhanced in CA1 pyramidal neuron spines and dendrites
from EST-treated slices. This result is consistent with the observation
of increased spine and synapse density and I/O slope of AMPA and NMDA
currents. Consistently, increased NMDA binding sites (Weiland
1992), and protein levels of NMDAR1 (Gazzaley et al.
1996
), associated with increased spine/synapse density
and sensitivity of CA1 cells to NMDA-dependent synaptic input
(Woolley et al. 1997
) have been reported in the
hippocampus of EST-treated OVX animals. However, potential effects of
EST on voltage-gated Ca2+ channel activity cannot be
excluded. The possibility of differences in Ca2+ buffering
and/or sequestration could reflect another potential effect of
estradiol as a growth factor, namely the modulation of dendritic
organelles functioning as intracellular Ca2+ stores and/or
sinks, such as the smooth endoplasmic reticulum (Pozzo-Miller et
al. 1997
).
The larger Ca2+ transients in dendritic shafts mediated by
synaptic activation of NMDA receptors in EST-treated slices observed in
our study are likely due to enhanced NMDA-mediated Ca2+
influx into dendritic compartments from a higher density of spines expressing functional Ca2+-permeable NMDA receptors. The
higher Ca2+ levels reached within spines in EST-treated
slices could represent attenuated diffusion from spine heads into
dendritic shafts due to the higher Ca2+ concentration of
the latter compartment brought up from a higher density of spines with
Ca2+-permeable NMDA receptors. Alternatively,
estradiol-induced spines may express a higher density of NMDA receptors
in their postsynaptic densities, or these receptors may have larger
Ca2+ permeability or single-channel properties (but see
Wong and Moss 1994). Either enhanced Ca2+
release from intracellular stores (Pozzo-Miller et al.
1996
), or impaired Ca2+ sequestration and/or
buffering, may also contribute to enhanced spine Ca2+
elevations in EST-treated slices.
An increase in plasma membrane surface area by the addition of
new dendritic spines would induce a decrease in cell input resistance
(Rn). Woolley et al. (1997)
reported that there were no significant differences in
Rn between CA1 cells from OVX and EST-treated
OVX rats, although a negative correlation between spine density and
Rn was statistically significant. Since in that study (Woolley et al. 1997
) the spine density increased
by only 22-28% in EST-treated OVX rats, we conceive that the much
larger increases in spine density observed in the EST-treated slice
cultures (almost 100%) could account for the statistically significant differences in Rn in our study. The contribution
of increased dendritic length to reduced Rn was
not ruled out in our study, although this parameter was not affected by
EST in OVX rats (Woolley and McEwen 1994
). In addition,
the measurements of Rn were performed under
conditions in which background synaptic activity and its potential
contribution to Rn were completely eliminated.
CA1 neurons were voltage clamped at
80 mV in the presence of
nimodipine, picrotoxin, CNQX, and D,L-APV to block
high-voltage activated L-type Ca2+ channels,
GABAA, AMPA, and NMDA-type glutamate receptors,
respectively. Furthermore, patch pipettes were filled with
Cs+-based intracellular solution containing QX-314 to block
K+ and Na+ channels, as well as
GABAB receptor activity. The significant decrease in
Rn is therefore consistent with the observed
increase in membrane surface area provided by the addition of new
dendritic spines in CA1 neurons from EST-treated slices.
We have also provided evidence of higher density of synaptic contacts
by double immunofluorescent staining of synaptophysin and -CaMKII in
hippocampal slice cultures after EST treatment, reminiscent of similar
effects in primary cultures of embryonic hippocampal neurons, estimated
by immunofluorescence staining of synaptophysin (Murphy and
Segal 1996
) and synaptotagmin (Murphy, unpublished
observations). An increase of presynaptic release by EST could also
account for the observed enhancement of I/O curves and Ca2+
signaling and is consistent with the lower threshold for LTP induction
in EST-OVX and proestrous animals.
Estradiol has been implicated in cognitive function in both
experimental animal models (Luine 1994) and
postmenopausal women (Sherwin 1996
). Because NMDA
receptors and the hippocamal formation have been strongly linked to LTP
as a cellular model of learning and memory (Bliss and
Collingridge 1993
), estradiol may exert its effects in
cognitive functions via the induction of new dendritic spines with
enhanced NMDA-mediated Ca2+ signaling mechanisms.
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ACKNOWLEDGMENTS |
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We acknowledge Drs. Thomas S. Reese and S. Brian Andrews for support and comments on the manuscript, the Light Imaging Facility of the National Institute of Neurological Disorders and Stroke, National Institutes of Health, for use of the confocal laser scanning microscope, and David Ide (National Institute of Mental Health, NINDS Research Services Branch) for building the brain slicer.
This work was supported by the NIH Intramural Research Program.
Present address of D. D. Murphy: Dept. of Pathology and Laboratory Medicine, University of Pennsylvania, 3rd Floor Maloney, 3600 Spruce St., Philadelphia, PA 19104.
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FOOTNOTES |
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Present address and address for reprint requests: L. D. Pozzo-Miller, Dept. of Neurobiology, University of Alabama at Birmingham, CIRC 429, 1719 Sixth Ave. South, Birmingham, AL 35294.
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 4 June 1998; accepted in final form November 1998.
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REFERENCES |
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