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Volume 16, Number 13,
Issue of July 1, 1996
pp. 4059-4068
Copyright ©1996 Society for Neuroscience
Regulation of Dendritic Spine Density in Cultured Rat Hippocampal
Neurons by Steroid Hormones
Diane D. Murphy1 and
Menahem Segal2
1 Laboratory of Neurobiology, National Institute of
Neurological Disorders and Stroke, National Institutes of Health,
Bethesda, Maryland 20892, and 2 Department of Neurobiology,
The Weizmann Institute, Rehovot 76100, Israel
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
The effects of gonadal steroid hormones on dendritic spines were
studied in hippocampal neurons that were dissociated and grown in
culture for 2-3 weeks. Exposure to estradiol caused up to a twofold
increase in dendritic spine density in these neurons. The effect of
estradiol was stereospecific and blocked by the steroid antagonist
tamoxifen. The estradiol-induced rise in spine density was blocked by
the NMDA antagonist APV, but not by the AMPA/KA antagonist DNQX. The
estradiol-induced rise in spine density was blocked by the
serine/threonine kinase inhibitor H7, but not by the tyrosine kinase
inhibitor genestein, and was partially mimicked by PMA, an activator of
protein kinase C. Estradiol also caused an increase in the fluorescence
intensity of synaptophysin-immunoreactive terminals, corresponding to
presynaptic boutons. Finally, estradiol caused a rise in
[Ca]i reactivity of the cultured neurons to
topical application of glutamate. These studies are the first to
examine receptor and second messenger regulation of dendritic spines,
and they illustrate the viability of cultured neurons as a powerful
test system to address issues related to the regulation of dendritic
spine maturation.
Key words:
dendritic spines;
estradiol;
culture;
hippocampal
neurons;
calcium;
plasticity
INTRODUCTION
Dendritic spines are the loci of synaptic
interactions among central neurons. Their susceptibility to changes in
afferent stimulation makes dendritic spines the prime candidates for
serving the long-term morphological substrates of neuronal plasticity.
In the hippocampus, which has been associated with learning and memory,
spines may be essential for the induction, associativity, specificity,
and endurance of long-term potentiation (Harris and Kater, 1994 ).
Despite extensive interest in dendritic spines, progress toward
understanding the mechanisms governing their generation and
morphological plasticity has been rather slow. This is attributable
partly to their minute size, which is at the limit of optical
resolution, and their large density and heterogeneity in neurons of the
intact brain.
Dissociated cultures of hippocampal neurons offer a viable and
convenient means for studying the factors that regulate the development
and functions of dendritic spines. The two-dimensional nature of
cultured neurons, as well as the relative sparseness of dendritic
spines in culture, which are approximately one third to one fourth the
density of those neurons in vivo, makes their study
technically achievable (Papa et al., 1995; Segal, 1995a,b).
It has been observed recently that the density of dendritic spines in
the intact rat hippocampus undergoes marked variations during the
estrous cycle. A higher density of dendritic spines coincides with high
levels of estrogen (Woolley et al., 1990), and the addition of
estradiol to ovariectomized rats causes a marked increase in spine
density (Woolley and McEwen, 1994). Circulating estrogens are prevalent
during neonatal development and so may be active in neonatal spine
formation. In fact, estrogens may be regulating synaptic plasticity in
adult brains as well. The hippocampus possesses estrogen receptors, and
the brain is able to synthesize steroid hormones, independent of the
gonads or adrenals (Singh et al., 1994). This would suggest that
estrogens play a major role in neuronal activity, regulating not only
reproductive behavior but also neuronal plasticity. In the present
study, we measured the effects of steroid hormones on dendritic spine
density in cultured hippocampal neurons and began a systematic analysis
of the receptor types and second messenger mechanisms associated with
the effects of estradiol on spine density.
MATERIALS AND METHODS
Culture of hippocampal neurons. Hippocampal cultures
were prepared as described previously (Papa et al., 1995). Briefly,
19-d-old embryos were taken from anesthetized Wistar rats. Their brains
were removed and placed in ice-cold (4°C) L15 medium supplemented
with 0.6% glucose and 15 µg/ml gentamicin. The hippocampus was
dissected out and mechanically disaggregated by gentle trituration
using a Pasteur pipette. Dissociated cells (500,000 cells/well) were
plated onto 12 mm glass coverslips that were coated with
poly-L-lysine (15 µg/ml) and then sterilized by
ultraviolet light. The plating medium was Eagle's MEM containing 10%
heat-inactivated horse serum, 5% fetal calf serum, 2 mM glutamine, 0.6% glucose, and 15 µg/ml
gentamicin. Cells were incubated at 37°C with 8%
CO2. The first change of medium, ~4-6 d after
plating, included 50 µg/ml uridine and 20 µg/ml deoxyuridine to
prevent glial cell overgrowth; thereafter, the cultures were fed 1-2
times/week with Eagle's MEM and 10% horse serum.
Cell dosing. Experimental solutions were prepared in
hippocampal growth media on the day of dosing (14 d in
vitro) and placed in the incubator to reach the appropriate
temperature and pH. Half of the media was removed from wells and
replaced with experimental media to give the appropriate final desired
concentration. Unless indicated otherwise, 17- -estradiol was used at
the intermediate dose of 0.1 µg/ml. The estrogen receptor antagonist
tamoxifen was used at 1 µg/ml to ensure saturation of binding sites.
Other drugs that were used (all from Sigma, St. Louis, MO) included
 -estradiol (1 µg/ml), progesterone (125 µg/ml), 2-APV (50 µM), DNQX (50 µM),
tetrodotoxin (TTX) (1 µM),
1-(5-isoquinolinesulphonyl) 2-methylpiperazine hydrochloride (H7) (15 µM), phorbol 12-myristate 13-acetate (PMA) (150 nM), and genestein (20 µM). After 2-3 d in experimental solutions,
cells were washed with PBS and fixed for 30 min at room temperature
with 4% paraformaldehyde in PBS. In each of the experiments conducted
in this study, a control and an estradiol-treated culture were always
compared with the addition of the test drug in a 2 × 2 design.
Confocal imaging. Fixed cells were stained by pressure
ejection of microdrops of
1,1 -dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate
(DiI) dissolved in cod-liver oil (Hosokawa et al., 1992). Thirty to
forty isolated, medium-sized (15-25 µm soma diameter) cells were
labeled on each coverslip, and these were stored in PBS at 4°C for
~6 hr. Cells were visualized with a Zeiss confocal laser scanning
microscope (CLSM) (National Institute of Neurological Disorders and
Stroke Imaging Facility, National Institutes of Health) using a 100×,
1.4 NA oil-immersion objective. A single, low-power scan was made of
each individual cell soma, followed by four to eight serial, zoomed,
optical sections of three to four regions of secondary dendrites around
each cell. Individual dendrites were reconstructed three-dimensionally
from the optical serial sections using National Institutes of Health
Image software on a Power Macintosh. Individual spines were identified
clearly as short, 1-3 µm protrusions at a right angle from the
dendrite, some having a distinct spine head. These spines were
different from filopodia in that the latter were longer and thinner at
the limit of resolution. As seen before, filopodia were abundant in the
younger cultures (Papa et al., 1995). Spines were counted in 50 µm
segments of dendrites. In a typical experiment, ~2000-4000 spines
were counted on 200 dendritic segments in 50 neurons. Average spine
densities per 50 µm dendritic segments were then calculated for each
treatment group, along with the SE. Groups of spines were compared
using the Student's t test.
Synaptophysin (SF) immunoreactivity. Cultures were fixed in
4% paraformaldehyde at room temperature for 30 min. Cells were washed
three times in PBS and subsequently blocked with 10% horse serum and
0.2% saponin in PBS for 20 min at room temperature. The blocking
solution was shaken off, and the cells were incubated with rabbit
anti-human SF (Dako, Glostrup, Denmark) overnight at 4°C. The
following morning, cells were washed again in PBS and incubated with
fluorescently labeled goat anti-rabbit IgG at room temperature for 60 min. Cells were washed in PBS and mounted in Vectashield (Vector Labs).
Images of SF-immunoreactive nerve terminals were taken with the CLSM,
at the same magnifications as for the DiI-stained cells, and stored for
later analysis. The analysis involved automatic counting of
SF-immunostained nerve terminals and measuring their size and
fluorescence intensity, using National Institutes of Health image
software. This analysis was conducted on 25 fields of controls and 27 fields of estradiol-treated cultures.
Calcium imaging. Cells were loaded with 5 µM Fura-2 AM,
prepared from a stock DMSO solution of 1 mM
(Molecular Probes), and 1 µl pluronic acid, prepared from a stock
solution of 20 mg/ml DMSO. Dye loading was in a HEPES-buffered (10 mM) recording medium containing TTX (1 µM), Ca2+ (2 mM), Mg2+ (1 mM), NaCl (130 mM), KCl (4 mM), and glucose (10 mM).
The medium had a pH of 7.4, and the osmolarity was adjusted to 320 mOsm
with sucrose. The cultures were exposed to the dye for 45 min, followed
by a 30 min wash. They were then used for imaging of glutamate-evoked
[Ca]i changes. The culture coverslips were
glued to the bottom of a small, 0.5 ml flow chamber placed in an
inverted Zeiss microscope. The cells were imaged with a cooled CCD
camera (Photometrics PXL) linked to an IBM computer. Images were taken
at 5 sec intervals before and after exposure of the cells to pressure
application of 1 mM glutamate applied through a
micropipette with a tip diameter of 2-3 µm. The ratio of
fluorescence emitted from the cells to excitation wavelengths of 340 and 380 nm were calculated before and after exposure to glutamate. The
intensity of excitation with the two wavelengths was equated with
neutral density filters to yield a ratio of ~1 at resting
[Ca]i. Groups of cells were compared using
independent t tests and ANOVA wherever appropriate.
RESULTS
Effects of steroid hormones on dendritic spine density
Estradiol treatment for up to 3 d did not have an apparent effect
on soma and primary dendritic morphology. The size of the somata was
measured in 20 cells from each of the control and estradiol-treated
cultures. The mean was about the same in the two groups: 334.5 ± 46.3 µm2 for control and 352.2 ± 72.8 µm2 for estradiol-treated cells. Likewise,
dendritic diameters, measured randomly in the same regions where spines
were counted, were not different between controls (1.12 ± 0.07 µm;
mean ± SEM; n = 35 dendritic segments) and
estradiol-treated cultures (0.97 ± 0.045 µm; n = 35;
p > 0.05).
Spine density increased significantly after treatment of the
cultures with estradiol; in control cells, spine density was 8.91 ± 0.57 (mean ± SEM) spines per 50 µm dendritic segment (Figs.
1, 2), whereas in cells treated with 0.1 µg/ml 17-
estradiol, spine density was up to 20.44 ± 0.92 spines per 50 µm
segment. This effect was stereoselective and was not seen after
treatment of the cells with -estradiol. Likewise, progesterone did
not affect spine density in these cells, but it did block the action of
estradiol (Fig. 2).
Fig. 1.
Three-dimensional, reconstructed images of
DiI-stained cultured hippocampal neurons. A, B, Low-power
images of control (A) and estradiol-treated (B)
neurons. C, D, Zoomed images of dendrites of control
(C) and estradiol-treated (D) cultured neurons.
The counting of spines in these and similar dendrites requires going
back and forth between the individual planes and the reconstructed
image to verify that each and every spine is counted only once.
E-G, Additional zoomed images of estradiol-treated
(E), H7-treated (F), and combined
H7/estradiol-treated cultures (G). Note the appearance of
long filopodia-like spines (arrowheads) in the H7-treated
cultures. Scale bar: A, B, 12 µm; C, D, 6 µm;
E-G, 3 µm.
[View Larger Version of this Image (128K GIF file)]
Fig. 2.
Estradiol produces a stereoselective effect on
dendritic spines. 17--est (17-b est.) (0.1 µg/ml) and
not -est (a-est.) (1 µg/ml) causes a larger than
twofold increase in spine density. The effects of estradiol are not
mimicked by progesterone (125 µg/ml), which blocks the effects of
estradiol. In this and the following figures, ** indicates statistical
difference from control using the Student's t test at
p < 0.001; * indicates statistical difference of
p < 0.05; n = 110, the total number of dendritic
segments analyzed in this experiment.
[View Larger Version of this Image (35K GIF file)]
The dose dependency of estradiol was tested first by using three
different doses of the hormone: 0.01 µg/ml, 0.1 µg/ml, and 1 µg/ml. All three doses caused a significant, nearly twofold increase
in spine density (data not shown). Thereafter, all other experiments
were performed at the intermediate dose of 0.1 µg/ml.
The effect of 17- estradiol is mediated by activation of an estrogen
receptor. In the presence of the estrogen receptor antagonist
tamoxifen, the effects of estradiol on spine density were completely
blocked (Fig. 3), indicating that estradiol did not
exert its action via some nonspecific route, but it acts on a genuine
estrogen receptor. In fact, tamoxifen produced a slight but
nonsignificant reduction in spine density in the control condition,
indicating that the growth medium does not contain significant
concentrations of estrogen.
Fig. 3.
Estrogen receptor antagonist tamoxifen
(Tam.) blocks the effects of estradiol (Est.) on
spine density. Tamoxifen (1 µg/ml) blocks the effects of 0.1 µg/ml
estradiol (E+T), even when applied 12 or 24 hr after the
beginning of exposure to estradiol. n = 259 dendritic
segments.
[View Larger Version of this Image (40K GIF file)]
The time course of the effect of estradiol was studied in two
types of experiments. In the first, estradiol was kept in the culture
medium for up to 4 d. At various time periods, cells were fixed, and
the density of the spines was measured as before. In these experiments,
it seems that estradiol began to have a significant enhancing effect on
spine density within 48 hr of exposure to the drug (Fig.
4). An additional rise in spine density was seen for up
to 4 d after exposure to the drug. In the second series of experiments,
the cultures were exposed first to estradiol, followed by their
simultaneous exposure to tamoxifen for 12-24 hr (Fig. 3). With both
time points analyzed, the addition of tamoxifen reduced significantly
the action of estradiol. Thus, it seems that estradiol has to be
present for at least 24 hr to exert its action in the hippocampus.
Fig. 4.
Time course of estradiol action on dendritic spine
density. Estradiol (0.1 µg/ml) produces a significant effect on spine
density within 48 hr of its application. n = 240 dendritic
segments.
[View Larger Version of this Image (36K GIF file)]
Dendritic spines are the sites of excitatory synaptic connections and
are thus enriched in glutamate receptors. We therefore examined the
role of glutamate receptors in the action of estradiol on dendritic
spine density. For this we employed the NMDA receptor antagonist 2-APV
and the AMPA/KA receptor antagonist DNQX. 2-APV completely reversed the
action of estradiol on dendritic spine density (Fig. 5).
By contrast, DNQX had only a negligible effect on estradiol-induced
spine formation. These data indicate that the effects of estradiol are
mediated by the activation of an NMDA receptor. The possibility that
the activation of the NMDA receptor is an indirect effect resulting
from an increase in spontaneous activity of the neurons in the culture
was examined by blocking action-potential discharges using TTX. By
itself, TTX had no effect on spine density in the culture, but it
reduced the effects of estradiol by ~35% (Fig. 5). This effect was
smaller than that produced by 2-APV, but it indicates that an increase
in spontaneous action-potential discharges may play a role in the
effect of estradiol on spine formation.
Fig. 5.
The NMDA receptor antagonist APV blocks the
effects of estradiol on spine density. Estradiol (0.1 µg/ml) produces
a twofold increase in spine density. APV, which has no
effect of its own at 50 µM, blocks the effect
of estradiol. DNQX, an AMPA/KA antagonist, has only a
partial but a significant effect on estradiol action. Likewise,
TTX reduces significantly the effect of estradiol but does
not eliminate it. n = 264 dendritic segments.
[View Larger Version of this Image (52K GIF file)]
The involvement of second messenger systems in the action of estradiol
on spine formation was examined by comparing the effects of a drug that
blocks serine/threonine kinase (H7) with a drug that blocks tyrosine
kinase (genestein). H7 by itself had no effect on spine density, but it
caused the formation of new filopodia, defined as thin, long (>3
µm), headless appendages resembling headless spines in immature
cultures (Fig. 1) (Papa et al., 1995). In the presence of H7, the
effects of estradiol on spine density were blocked completely (Fig.
6). In contrast, genestein did not affect spine density,
and neither did it interact with the effects of estradiol. Thus, it
seems that estradiol acts via a serine/threonine kinase, possibly
protein kinase C (PKC), to enhance formation of new spines.
Fig. 6.
A serine/threonine kinase inhibitor, H7
(15 µg/ml), but not a tyrosine kinase inhibitor, genestein
(Gen.) (20 µg/ml), blocks the effect of estradiol on
dendritic spine density. The phorbol ester PMA (150 nM) has a direct enhancing action on spine
density but also reduces significantly the effect of estradiol.
n = 300 dendritic segments.
[View Larger Version of this Image (59K GIF file)]
To examine this hypothesis further, we exposed cells to the phorbol
ester PMA, an activator of PKC. After 30 hr of exposure to PMA, there
was a modest but significant increase in mean spine density from 9.0 ± 0.31 to 11.96 ± 0.42 spines per 50 µm segment (Fig. 6). In the same
experiment, estradiol caused a rise of spine density to 15.4 ± 0.46 spines per 50 µm dendrite. Interestingly, there was no additive
effect of the two drugs on spine density. In fact, the presence of PMA
significantly reduced the effect of estradiol to 12.0 ± 0.43 spines
per 50 µm segment.
Effects of estradiol on synapse formation
Estradiol may cause formation of new spines by converting existing
shaft synapses into spine synapses or by producing new synapses.
Alternatively, the new spines may be simple, nonfunctional extrusions
from the dendrites, having no presynaptic terminals attached to them.
To examine these possibilities, we stained control and
estradiol-treated cultures with SF antibodies. Analysis of bouton
staining in 25 and 27 random fields taken from control and
estradiol-treated cultures, respectively, revealed no difference in the
size of the terminals (32.1 ± 0.3 and 32.9 ± 0.3 pixels, where each
pixel is 0.125 µm × 0.125 µm, corresponding to an approximate area
of 0.6 µm2). There was, however, a significant
difference in the staining intensity per terminal: the controls were
76.5 ± 0.78 intensity units, compared with 102.7 ± 0.82 units in the
estradiol-treated cultures (p < 0.001) (Fig.
7). This may indicate an increased density of SF
protein, suggesting that the new spines may represent an enhanced
density of synaptic connections. It is still unclear whether all of the
newly formed spines contain functional synapses, or whether the
SF-immunoreactive boutons form multiple contacts with one or several
spines. This remains to be determined by additional experimentation
with high-power electron microscopy.
Fig. 7.
SF-containing boutons are different between
control (A) and estradiol-treated (B) cultures.
The intensity of staining is higher in the estradiol-treated cultures.
Consult text for details. Scale bar, 10 µm.
[View Larger Version of this Image (90K GIF file)]
Imaging of calcium variations in estradiol-treated cultures
The association of estradiol effects on spine formation with
activation of the NMDA receptor led us to examine the effects of
estradiol on the reactivity of cultured hippocampal neurons to
glutamate. Two types of experiments were conducted. In the first, we
studied the acute effects of estradiol on reactivity to glutamate in
drug-naive cultures; in the second, we compared control and
estradiol-treated cultures for reactivity to glutamate acting on NMDA
and non-NMDA receptors. If indeed estradiol interacts directly with the
NMDA receptor, one can expect that exposure to estradiol will modify
reactivity to glutamate. The magnitude of the response and the time
course of the recovery from calcium load after exposure to glutamate
were compared before and after superfusion of cells with 1 µM estradiol. Exposure to glutamate caused a
transient rise of [Ca]i in all cells examined
to a level of ~300 nM, as estimated from the
ratio of emitted fluorescence to 340/380 nm illumination. This response
peaked within 5 sec after the onset of exposure to glutamate and
recovered to near basal levels within the following 10 sec. Exposure to
estradiol caused an initial small and insignificant decline in
reactivity to glutamate (Fig. 8B). No further
decrease in reactivity to glutamate was seen with extended exposure to
estradiol.
Fig. 8.
Intracellular calcium
([Ca]i) changes after exposure to pulse
application of glutamate. A, An illustration of a ratioed
image of a Fura-2-loaded cell at rest (left) and after
exposure to glutamate (right). The measurements in this and
the following figure were made of the region in the center of the cell,
~15 µm in diameter, which includes the nucleus. B, Lack
of acute effects of estradiol (0.1 µM) on
reactivity to successive application of glutamate (Glu). For
each sequence, a baseline control value was measured, followed by puff
application of glutamate (arrowhead) followed by three
measurements of fluorescence, taken at 5 sec intervals. Five to ten
minutes elapsed between successive applications of glutamate. Note that
at the peak response to glutamate, the heterogeneity (i.e., the SEM) is
smaller than during recovery, indicating that the rate of recovery is
widely distributed among different cells. C, A comparison of
responses to glutamate of control (Ct) and estradiol-treated
(Est) cultured neurons. A significant difference between the
two groups in both the peak and the recovery of rest
[Ca]i is evident.
[View Larger Version of this Image (31K GIF file)]
In the other series of experiments, reactivity to glutamate was
compared between the estradiol-treated and control cultures; 82 estradiol-treated cells exhibited a significantly larger (by 23%) and
more persistent response to glutamate than 48 control cells did (Fig.
8C).
In an attempt to dissociate between NMDA- and non-NMDA- (primarily
voltage-gated calcium channels activated by glutamate-induced
depolarization) mediated calcium changes, we repeated this study using
conditions that favor activation of one of the two types of receptors
(Fig. 9). As seen in the previous experiment,
estradiol-treated cells (n = 24) exhibited a significantly
larger (by 33%) response to glutamate than control cells did
(n = 49). The cells were then washed with normal medium
containing 0 mM Mg2+,
glycine (10 µM), and a
Ca2+ channel blocker, verapamil (20 µM). Under these conditions, which favor
activation of NMDA receptors, estradiol-treated (n = 34)
cells exhibited an even larger (by 95%) response to glutamate than
control (n = 19) cells did. The cultures were then washed
with normal medium containing 50 µM 2-APV.
Under these conditions, the responses to glutamate, which now activates
only non-NMDA receptors, was larger by 80% in estradiol-treated cells
(n = 31) than in control cells (n = 31). These
experiments indicate that estradiol causes an increase in reactivity of
chronically exposed neurons to glutamate. Whether this is caused by an
increase in the density of glutamate receptors, by a change in receptor
affinity to glutamate, or by an increase in some second messenger
systems associated with the glutamate receptors remains to be
determined. A correlation seems to exist between the effects of
estradiol on cellular morphology (i.e., spine formation) and function
(glutamate responses).
Fig. 9.
Effects of estradiol (Est) on
[Ca]i reactivity of neurons to topical
application of glutamate in conditions that favor activation of NMDA-
and non-NMDA-linked [Ca]i changes.
A, The total response to glutamate under normal conditions,
as in Figure 8C, are seen. B, The culture medium
contains nominally zero Mg2+, glycine, and a
voltage-gated calcium Ca2+ blocker, verapamil
(see Results for further details), to allow activation of NMDA receptor
type. C, The cultures were treated with normal medium
containing 1 mM Mg2+, to
which 50 µM APV was added to allow detection of
non-NMDA receptor/channels. In both B and C, a
marked difference between normal and estradiol-treated cultures is
seen.
[View Larger Version of this Image (14K GIF file)]
DISCUSSION
Throughout the past decade, studies have shown the involvement of
steroid hormones in the CNS (for review, see McEwen et al., 1993).
In vivo models have explored the effects of estradiol in the
hippocampus. Ovariectomized adult rats display a dramatic decrease in
the density of dendritic spines, which can be reversed by the
replacement of estradiol (Gould et al., 1990), and supplemental
estradiol treatment can increase the density of dendritic spines. It
has also been shown that normal fluctuations of estradiol during the
5-d estrous cycle in rats causes fluctuations in dendritic spines
(Woolley et al., 1990) as well as in the density of synapses in the
hippocampus (Woolley and McEwen, 1993). These effects seem to be
mediated by an NMDA receptor-dependent mechanism (Woolley and McEwen,
1994). Behavioral studies have demonstrated that ovariectomized adult
rats displayed impaired learning and memory, which was reversible with
estradiol replacement (Singh et al., 1994). Clearly, estradiol has a
marked effect on the modulation of spines in vivo and may be
related to the effect of estradiol on hippocampal functions in the
intact brain.
In the present study, we have successfully reproduced the in
vivo estradiol response in a tissue culture model of hippocampal
neurons. Our results are similar to those seen in vivo:
17--estradiol significantly increased the density of spines on
hippocampal neurons. This effect is seen 48 hr, not 24 hr, after the
onset of estradiol treatment, which is the same time course seen
in vivo (Woolley and McEwen, 1993).
The cells that we analyzed are most likely medium-size CA1 pyramidal
neurons, as the larger CA3 pyramids do not survive well in our culture
conditions, and we were unable to detect the large mossy fiber-related
dendritic spines typical of these neurons. Also the smaller, granular
cells of the dentate gyrus have a distinctly different morphology and
tend to produce clumps of cells, which were not studied herein. CA1
cells were those studied in vivo. One major difference
between the in vivo and in vitro CA1 cells is the
density of their dendritic spines, which are up to 8-10 times higher
in vivo than in vitro (Harris et al., 1992). This
difference can be caused by the relatively small number of afferents
impinging on the dendrites of the in vitro cell, by the
growth conditions, and by the possible lack of intrinsic growth
factors. Additionally, the cultures are studied in a juvenile state,
2-3 weeks in vitro, corresponding to 10-17 d in neonatal
rats. Despite the diminished number of spines, it is easier and more
reliable to study dendritic spine density in the isolated culture dish,
where each spine can be counted and the spine density analysis is fast
and unequivocal. The initial low density of spines in the culture may
underlie the larger effect of estradiol seen here by comparison with
the in vivo case, where the reported changes are
30-50%.
The in vitro controlled tissue culture conditions
allow us to study the mechanisms of estradiol action in relative
isolation and to begin analysis of the factors involved in dendritic
spine formation. The fact that the estradiol response can be blocked by
tamoxifen and that -estradiol does not mimic the effect of
-estradiol suggest that -estradiol binds specifically to the
estrogen receptor. The mechanism of action of estradiol may involve
activation of PKC, as seen elsewhere. Estradiol upregulates PKC in
ovaries and pituitary of several mammalian species, and this
upregulation is inhibited by tamoxifen (Hahnel and Gschwendt, 1995). If
estradiol activates PKC in hippocampal neurons, this may indicate that
spine formation is initiated at the molecular level by this second
messenger system. Indeed, the effect of estradiol is blocked by the
broad-band serine/threonine kinase inhibitor H7, indicating that a
kinase is involved in this action of estradiol. We chose H7 rather than
a more selective PKC or other kinase antagonist to maximize our ability
to observe some blocking effects on the action of estradiol. This
should not be taken as direct evidence that PKC is the specific kinase
associated with the action of estradiol and that more specific
antagonists should be used in the context. The present results
encourage such a search, in that they suggest that a serine/threonine
but not a tyrosine kinase is activated by estradiol to increase
dendritic spine density.
The role of the NMDA receptor in the action of estradiol is not
entirely clear. Activation of the NMDA receptor is crucial for the
induction of long-term potentiation in some synapses in the hippocampus
and elsewhere (Bliss and Collingridge, 1993). If spine formation is
associated with long-term plasticity in the brain, it will be
intuitively associated with activation of the NMDA receptor. Is
stimulation of kinases by estradiol causing the activation of the NMDA
receptor, which forms new spines, or does estradiol affect NMDA
receptors directly, which stimulates kinases to form new spines? Also,
how is the rather gradual (48 hr) response to estradiol related to the
fast action of glutamate at the NMDA receptor? These and related
questions await further experimentation. Estradiol is also likely to
increase spine density by affecting spontaneous synaptic activity of
cells in culture. Blockade of action-potential discharges with TTX
caused a partial blockade of the effects of estradiol on spine density.
This indicates that estradiol may cause the formation of new synapses
by increasing network activity, probably by increasing NMDA-mediated
synaptic potentials.
The estrogen-initiated large fluctuations in dendritic spine
density in the hippocampus, a structure traditionally associated with
cognitive processes in the brain, have important theoretical
implications with respect to the roles of dendritic spines in synaptic
integration. If the spine is the site of memory storage, i.e.,
modification in spine shape/density represents the means by which a
long-term change in synaptic efficacy is maintained, how can these
large but transient changes in spine density be related to long-term
memory storage? In fact, the estradiol effects may indicate just the
opposite: that spines may also serve transient roles related to changes
in synaptic strength occurring, for example, during the estrous cycle.
The more enduring changes associated with long-term memory may not be
seen in the hippocampus, or they may be associated with other
morphological or nonmorphological changes in neurons. These may include
receptor clustering, change in synaptic contact area, or spine form or
shape (Fifkova and Van Harreveld, 1977; Lee et al., 1980; Chang and
Greenough, 1984). The idea that spine density is not a vehicle for
long-term memory storage is not consistent with other recent
observations (Moser et al., 1994), and it is possible that spines have
multiple functions in the brain that encompass long-term and short-term
plasticity.
If not solely memory devices, what other functions can spines serve? It
has been suggested recently that intracellular calcium concentrations
can be regulated independent of the parent dendrites (Andrews et al.,
1988; Wickens, 1988; Guthrie et al., 1991; Gold and Bear, 1994; Segal,
1995a). Thus, dendritic spines may prevent calcium surges from
spreading into the parent dendrite and in that respect can be
considered neuroprotectants (Harris and Kater, 1994; Segal, 1995b).
Dendritic spines proliferate in the presence of excessive synaptic
activity (Annis et al., 1994; Bundman et al., 1994; Papa and Segal,
1996), although some reports claim the opposite (Muller et al., 1993;
Rocha and Sur, 1995). It is thought that most spines have one
excitatory synapse on the head (Harris et al., 1992) and that most, if
not all, excitatory synapses are on spines. Thus the doubling of spines
in response to estradiol represents a doubling of excitatory synaptic
inputs on each single spiny neuron. These dendritic spines can be
absorbed back to their parent dendrites when the need to deal with this
excessive synaptic activity is reduced. Our present observations are
congruent with this hypothesis.
When imaging calcium transients in response to glutamate stimulation,
we found that there was virtually no difference between the control
responses and those seen during acute exposure to estradiol. Cells that
had been treated with estradiol chronically (several days in culture),
however, displayed intracellular calcium transients that were larger
and longer in duration than those in controls. These findings would
imply that the morphological change in neurons, i.e., the increase in
spine density, may be responsible for the enhanced reactivity to
glutamate. We cannot rule out the alternative possibility of a genomic
action of estradiol on the glutamate receptors unrelated to
morphological changes. Further analysis is needed to determine the
causality between spine density and glutamate-evoked
[Ca]i.
The present results illustrate the viability of the culture as a model
system for the analysis of factors affecting the production of
dendritic spines in central neurons. The ability to control the
microenvironment of the cells and to expose the cells to drugs that may
have adverse effects in vivo, as well as the relative
simplicity of the cultured neurons, allows rapid progress in the study
of the mechanisms associated with dendritic spines.
FOOTNOTES
Received Dec. 8, 1995; revised April 2, 1996; accepted April 4, 1996.
We thank Drs. T. S. Reese and S. B. Andrews for their support and Dr.
C. Collin for help with the calcium imaging system.
Correspondence should be addressed to M. Segal, Department of
Neurobiology, The Weizmann Institute, Rehovot 76100, Israel.
REFERENCES
-
Andrews SB,
Leapman RD,
Landis DMD,
Reese TS
(1988)
Activity dependent accumulation of calcium in
Purkinje cell dendritic spines.
Proc Natl Acad Sci USA
85:1682-1685 .
[Abstract/Free Full Text]
-
Annis CM,
O'Dowd DK,
Robertson RT
(1994)
Activity-dependent
regulation of dendritic spine density on cortical pyramidal neurons in
organotypic slice culture.
J Neurobiol
25:1483-1493 .
[Web of Science][Medline]
-
Bliss TVP,
Collingridge GL
(1993)
A synaptic model of memory:
long term potentiation in the hippocampus.
Nature
361:31-39.
[Medline]
-
Bundman MC,
Pico RM,
Gall CM
(1994)
Ultrastructural
plasticity of the dentate gyrus granule cells following recurrent
limbic seizures: increase in somatic spines.
Hippocampus
4:601-610 .
[Web of Science][Medline]
-
Chang F,
Greenough WT
(1984)
Transient and enduring
morphological correlates of synaptic activity and efficacy in the rat
hippocampal slice.
Brain Res
309:35-46 .
[Web of Science][Medline]
-
Fifkova E,
Van Harreveld A
(1977)
Long lasting morphological
changes in dendritic spines of dentate granular cells following
stimulation of the entorhinal area.
J Neurocytol
6:211-230 .
[Web of Science][Medline]
-
Gold JI,
Bear MF
(1994)
A model of dendritic spine
Ca2+ concentration exploring possible bases for a
sliding synaptic modification threshold.
Proc Natl Acad Sci USA
91:3941-3945 .
[Abstract/Free Full Text]
-
Gould E, Woolley C, Frankfurt M, McEwen B (1990) Gonadal
steroids regulate dendritic spine density in hippocampal pyramidal
cells in adulthood. J Neurosci 10:1286-1291.
-
Guthrie PB,
Segal M,
Kater SB
(1991)
Independent regulation
of calcium revealed by imaging dendritic spines.
Nature
354:76-80 .
[Medline]
-
Hahnel R,
Gschwendt M
(1995)
Interaction between protein
kinase C (PKC) and estrogens.
Int J Oncol
7:11-16.
-
Harris KM,
Kater SB
(1994)
Dendritic spines: cellular
specializations imparting both stability and flexibility to synaptic
function.
Annu Rev Neurosci
17:341-371 .
[Web of Science][Medline]
-
Harris KM,
Jensen FE,
Tsao BH
(1992)
Three-dimensional
structure of dendritic spines and synapses in rat hippocampus (CA1) at
postnatal day 15 and adult ages: implications for the maturation of
synaptic physiology and long-term potentiation.
J Neurosci
12:2685-2705 .
[Abstract]
-
Hosokawa T,
Bliss TVP,
Fine A
(1992)
Persistence of
individual dendritic spines in living brain slices.
NeuroReport
3:477-480 .
[Web of Science][Medline]
-
Lee KS,
Schottler F,
Oliver M,
Lynch G
(1980)
Brief bursts of
high-frequency stimulation produce two types of structural change in
the rat hippocampus.
J Neurophysiol
44:247-258 .
[Free Full Text]
-
McEwen BS,
Cameron H,
Chao H,
Gould E,
Magarinos A,
Watanabe Y,
Woolley C
(1993)
Adrenal steroids and plasticity of hippocampal
neurons: toward an understanding of underlying cellular and molecular
mechanisms.
Cell Mol Neurobiol
13:457-482 .
[Web of Science][Medline]
-
Moser MB,
Trommald M,
Andersen P
(1994)
An increase in
dendritic spine density on hippocampal CA1 cells following
spatial-learning in adult rats suggests the formation of new synapses.
Proc Natl Acad Sci USA
91:12673-12675 .
[Abstract/Free Full Text]
-
Muller M,
Gahwiler BH,
Rietschin L,
Thompson SM
(1993)
Reversible loss of dendritic spines and altered
excitability after chronic epilepsy in hippocampal slice cultures.
Proc Natl Acad Sci USA
90:257-261 .
[Abstract/Free Full Text]
-
Papa M, Segal M (1996) Morphological plasticity in dendritic
spines of cultured hippocampal neurons. Neuroscience,
71:1005-1011.
-
Papa M,
Bundman MC,
Greenberger V,
Segal M
(1995)
Morphological analysis of dendritic spine
development in primary cultures of hippocampal neurons.
J Neurosci
15:1-11 .
[Abstract]
-
Rocha M,
Sur M
(1995)
Rapid acquisition of dendritic spines
by visual thalamic neurons after blockade of
N-methyl-d-aspartate receptors.
Proc Natl Acad Sci USA
92:8026-8030 .
[Abstract/Free Full Text]
-
Segal M
(1995a)
Imaging of calcium variations in living
dendritic spines of cultured rat hippocampal neurons.
J Physiol (Lond)
486:285-296.
-
Segal M
(1995b)
Spines for neuroprotection: a hypothesis.
Trends Neurosci
11:468-471.
-
Singh M,
Meyer E,
Millard W,
Simpkins J
(1994)
Ovarian
steroid deprivation results in a reversible learning impairment and
compromised cholinergic function in female Sprague-Dawley rats.
Brain Res
644:305-312 .
[Web of Science][Medline]
-
Wickens J
(1988)
Electrically coupled but chemically isolated
synapses: dendritic spines and calcium in a rule for synaptic
modification.
Prog Neurobiol
31:507-528 .
[Web of Science][Medline]
-
Woolley C,
McEwen B
(1993)
Roles of estradiol and
progesterone in regulation of hippocampal dendritic spine density
during the estrous cycle in the rat.
J Comp Neurol
336:293-306 .
[Web of Science][Medline]
-
Woolley C,
McEwen B
(1994)
Estradiol regulates hippocampal
dendritic spine density via an
N-methyl-d-aspartate receptor
dependent mechanism.
J Neurosci
14:7680-7687 .
[Abstract]
-
Woolley CS,
Gould E,
Frankfurt M,
McEwen BS
(1990)
Naturally
occurring fluctuation in dendritic spine density on adult hippocampal
pyramidal neurons.
J Neurosci
10:4035-4039 .
[Abstract]
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|
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|
|
|
|
|
|
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|
|
|
|
|
|
|
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24(26):
5913 - 5921.
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|
|
|
|
|
|
|
C. Li, W. G. Brake, R. D. Romeo, J. C. Dunlop, M. Gordon, R. Buzescu, A. M. Magarinos, P. B. Allen, P. Greengard, V. Luine, et al.
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|
|
|
|
|
|
|
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|
|
|
|
|
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|
|
|
|
|
|
|
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|
|
|
|
|
|
|
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|
|
|
|
|
|
|
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[PDF]
|
|
|
|
|
|
|
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17(5):
831 - 844.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. N. Rudick and C. S. Woolley
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January 1, 2003;
144(1):
179 - 187.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. K. Amateau and M. M. McCarthy
A Novel Mechanism of Dendritic Spine Plasticity Involving Estradiol Induction of Prostaglandin-E2
J. Neurosci.,
October 1, 2002;
22(19):
8586 - 8596.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. A. DeFazio and S. M. Moenter
Estradiol Feedback Alters Potassium Currents and Firing Properties of Gonadotropin-Releasing Hormone Neurons
Mol. Endocrinol.,
October 1, 2002;
16(10):
2255 - 2265.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. M. Fillit
The Role of Hormone Replacement Therapy in the Prevention of Alzheimer Disease
Arch Intern Med,
September 23, 2002;
162(17):
1934 - 1942.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Zhao, Q. Chen, and R. D. Brinton
Neuroprotective and Neurotrophic Efficacy of Phytoestrogens in Cultured Hippocampal Neurons
Experimental Biology and Medicine,
July 1, 2002;
227(7):
509 - 519.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. W. Huntley, O. Gil, and O. Bozdagi
The Cadherin Family of Cell Adhesion Molecules: Multiple Roles in Synaptic Plasticity
Neuroscientist,
June 1, 2002;
8(3):
221 - 233.
[Abstract]
[PDF]
|
|
|
|
|
|
|
T. Jover, H. Tanaka, A. Calderone, K. Oguro, M. V. L. Bennett, A. M. Etgen, and R. S. Zukin
Estrogen Protects against Global Ischemia-Induced Neuronal Death and Prevents Activation of Apoptotic Signaling Cascades in the Hippocampal CA1
J. Neurosci.,
March 15, 2002;
22(6):
2115 - 2124.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. McEwen
Estrogen Actions Throughout the Brain
Recent Prog. Horm. Res.,
January 1, 2002;
57(1):
357 - 384.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. S. McEwen
Genome and Hormones: Gender Differences in Physiology: Invited Review: Estrogens effects on the brain: multiple sites and molecular mechanisms
J Appl Physiol,
December 1, 2001;
91(6):
2785 - 2801.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. A. Stafstrom-Davis, C. C. Ouimet, J. Feng, P. B. Allen, P. Greengard, and T. A. Houpt
Impaired Conditioned Taste Aversion Learning in Spinophilin Knockout Mice
Learn. Mem.,
September 1, 2001;
8(5):
272 - 278.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. N. Rudick and C. S. Woolley
Estrogen Regulates Functional Inhibition of Hippocampal CA1 Pyramidal Cells in the Adult Female Rat
J. Neurosci.,
September 1, 2001;
21(17):
6532 - 6543.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. M. Daniel and G. P. Dohanich
Acetylcholine Mediates the Estrogen-Induced Increase in NMDA Receptor Binding in CA1 of the Hippocampus and the Associated Improvement in Working Memory
J. Neurosci.,
September 1, 2001;
21(17):
6949 - 6956.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Korkotian and M. Segal
Regulation of Dendritic Spine Motility in Cultured Hippocampal Neurons
J. Neurosci.,
August 15, 2001;
21(16):
6115 - 6124.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. M. Adams, R. A. Shah, W. G. M. Janssen, and J. H. Morrison
Different modes of hippocampal plasticity in response to estrogen in young and aged female rats
PNAS,
June 20, 2001;
(2001)
141215898.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. McEwen, K. Akama, S. Alves, W. G. Brake, K. Bulloch, S. Lee, C. Li, G. Yuen, and T. A. Milner
Tracking the estrogen receptor in neurons: Implications for estrogen-induced synapse formation
PNAS,
June 19, 2001;
98(13):
7093 - 7100.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. D. Brinton
Cellular and Molecular Mechanisms of Estrogen Regulation of Memory Function and Neuroprotection Against Alzheimer's Disease: Recent Insights and Remaining Challenges
Learn. Mem.,
May 1, 2001;
8(3):
121 - 133.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. B. Dubal and P. M. Wise
Neuroprotective Effects of Estradiol in Middle-Aged Female Rats
Endocrinology,
January 1, 2001;
142(1):
43 - 48.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Goldin, M. Segal, and E. Avignone
Functional Plasticity Triggers Formation and Pruning of Dendritic Spines in Cultured Hippocampal Networks
J. Neurosci.,
January 1, 2001;
21(1):
186 - 193.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Braun and M. Segal
FMRP Involvement in Formation of Synapses among Cultured Hippocampal Neurons
Cereb Cortex,
October 1, 2000;
10(10):
1045 - 1052.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Feng, Z. Yan, A. Ferreira, K. Tomizawa, J. A. Liauw, M. Zhuo, P. B. Allen, C. C. Ouimet, and P. Greengard
Spinophilin regulates the formation and function of dendritic spines
PNAS,
August 1, 2000;
97(16):
9287 - 9292.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Hasbani, S. M. Underhill, G. De Erausquin, and M. P. Goldberg
Synapse Loss and Regeneration: A Mechanism for Functional Decline and Recovery after Cerebral Ischemia?
Neuroscientist,
April 1, 2000;
6(2):
110 - 119.
[Abstract]
[PDF]
|
|
|
|
|
|
|
L. H. Calizo and L. M. Flanagan-Cato
Estrogen Selectively Regulates Spine Density within the Dendritic Arbor of Rat Ventromedial Hypothalamic Neurons
J. Neurosci.,
February 15, 2000;
20(4):
1589 - 1596.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. L. Ross, D. Roeltgen, P. Feuillan, H. Kushner, and G. B. Cutler Jr.
Use of estrogen in young girls with Turner syndrome: Effects on memory
Neurology,
January 11, 2000;
54(1):
164 - 164.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Patrone, S. Andersson, L. Korhonen, and D. Lindholm
Estrogen receptor-dependent regulation of sensory neuron survival in developing dorsal root ganglion
PNAS,
September 14, 1999;
96(19):
10905 - 10910.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. M. Wise, M. J. Smith, D. B. Dubal, M. E. Wilson, K. M. Krajnak, and K. L. Rosewell
Neuroendocrine Influences and Repercussions of the Menopause
Endocr. Rev.,
June 1, 1999;
20(3):
243 - 248.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
B. S. McEwen and S. E. Alves
Estrogen Actions in the Central Nervous System
Endocr. Rev.,
June 1, 1999;
20(3):
279 - 307.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
B. S. McEwen
The Molecular and Neuroanatomical Basis for Estrogen Effects in the Central Nervous System
J. Clin. Endocrinol. Metab.,
June 1, 1999;
84(6):
1790 - 1797.
[Full Text]
|
|
|
|
|
|
|
L. D. Pozzo-Miller, T. Inoue, and D. D. Murphy
Estradiol Increases Spine Density and NMDA-Dependent Ca2+ Transients in Spines of CA1 Pyramidal Neurons From Hippocampal Slices
J Neurophysiol,
March 1, 1999;
81(3):
1404 - 1411.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. R. Foy, J. Xu, X. Xie, R. D. Brinton, R. F. Thompson, and T. W. Berger
17beta -Estradiol Enhances NMDA Receptor-Mediated EPSPs and Long-Term Potentiation
J Neurophysiol,
February 1, 1999;
81(2):
925 - 929.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Kraft, R. B. Levine, and L. L. Restifo
The Steroid Hormone 20-Hydroxyecdysone Enhances Neurite Growth of Drosophila Mushroom Body Neurons Isolated during Metamorphosis
J. Neurosci.,
November 1, 1998;
18(21):
8886 - 8899.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. D. Murphy, N. B. Cole, and M. Segal
Brain-derived neurotrophic factor mediates estradiol-induced dendritic spine formation in hippocampal neurons
PNAS,
September 15, 1998;
95(19):
11412 - 11417.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. M. Espreafico, D. E. Coling, V. Tsakraklides, K. Krogh, J. S. Wolenski, G. Kalinec, and B. Kachar
Localization of myosin-V in the centrosome
PNAS,
July 21, 1998;
95(15):
8636 - 8641.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. D. Murphy, N. B. Cole, V. Greenberger, and M. Segal
Estradiol Increases Dendritic Spine Density by Reducing GABA Neurotransmission in Hippocampal Neurons
J. Neurosci.,
April 1, 1998;
18(7):
2550 - 2559.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. E. Wood and T. J. Shors
Stress facilitates classical conditioning in males, but impairs classical conditioning in females through activational effects of ovarian hormones
PNAS,
March 31, 1998;
95(7):
4066 - 4071.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. S. Woolley, N. G. Weiland, B. S. McEwen, and P. A. Schwartzkroin
Estradiol Increases the Sensitivity of Hippocampal CA1 Pyramidal Cells to NMDA Receptor-Mediated Synaptic Input: Correlation with Dendritic Spine Density
J. Neurosci.,
March 1, 1997;
17(5):
1848 - 1859.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. D. Murphy and M. Segal
Morphological plasticity of dendritic spines in central neurons is mediated by activation of cAMP response element binding protein
PNAS,
February 18, 1997;
94(4):
1482 - 1487.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. M. Adams, R. A. Shah, W. G. M. Janssen, and J. H. Morrison
Different modes of hippocampal plasticity in response to estrogen in young and aged female rats
PNAS,
July 3, 2001;
98(14):
8071 - 8076.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
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