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The Journal of Neuroscience, February 15, 2000, 20(4):1589-1596
Estrogen Selectively Regulates Spine Density within the Dendritic
Arbor of Rat Ventromedial Hypothalamic Neurons
Lyngine H.
Calizo1 and
Loretta M.
Flanagan-Cato2
1 Institute of Neurological Sciences and
2 Department of Psychology, University of Pennsylvania,
Philadelphia, Pennsylvania 19104-6074
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ABSTRACT |
Estrogen acts in the hypothalamic ventromedial nucleus (VMH) to
promote female sexual behavior. One potential mechanism through which
estrogen may facilitate this behavior is by reconfiguring synaptic
connections within the VMH. Estrogen treatment increases the number of
synapses and dendritic spines in the VMH, but how this remodeling
occurs within the context of the local, behaviorally relevant
microcircuitry is unknown. The goal of this study was to localize
estrogen-induced changes in spine density within the VMH and relate
these to dendritic morphology and the presence of nuclear estrogen
receptor. The hypothalami from ovariectomized rats, treated with either
vehicle or estradiol, were lightly fixed, and VMH neurons were
iontophoretically filled with Lucifer yellow. Confocal microscopy was
used to examine neuronal morphology. Estrogen treatment increased
dendritic spine density by 48% in the ventrolateral VMH but had no
effect on spine density in the dorsal VMH. The primary dendrites of VMH
neurons were differentially affected by estrogen. Estrogen treatment
increased spine density twofold on the short primary dendrites but did
not affect spine density on long primary dendrites. Immunocytochemical
staining showed that none of the filled neurons expressed estrogen
receptor- . Thus, although the effect of estrogen on spine density is
localized to a VMH subdivision where estrogen receptor is expressed,
estrogen treatment induces spines on neurons that lack estrogen
receptor. Taken together, our results suggest that the effect of
estrogen on ventrolateral VMH spines is selective within the dendritic arbor of a neuron and may be mediated by an indirect, possibly transynaptic, mechanism.
Key words:
dendritic spines; estrogen; female sexual behavior; lordosis; Lucifer yellow cell filling; VMH
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INTRODUCTION |
A major goal of behavioral
neuroscience is to elucidate the neural basis of motivated behaviors.
Female rat sexual behavior has become a valuable model system, in part
because the macrocircuitry and hormonal influences are well described
(Pfaff et al., 1994 ). The actions of estrogen in the ventromedial
nucleus of the hypothalamus (VMH) are crucial to the promotion of this
behavior, as demonstrated by lesion, local infusion, and electrical
stimulation experiments (Mathews and Edwards, 1977a,b; Pfaff and
Sakuma, 1979a,b; Davis et al., 1982; Pleim et al., 1989). Additionally,
receptor autoradiography, immunocytochemistry, and in situ
hybridization have demonstrated the presence of estrogen receptors in
the VMH (Pfaff and Keiner, 1973; Simerly et al., 1990; DonCarlos et
al., 1991). Moreover, transneuronal tracing studies have confirmed the
serial connectivity of the VMH to the lumbar epaxial muscles that
execute the stereotypic female reproductive posture, lordosis (Daniels
et al., 1999). Nevertheless, key questions remain about the
microcircuitry within the VMH and how estrogen may alter neural
connectivity to control the expression of this behavior.
Previous Golgi studies have described the morphology of VMH neurons,
which have two to three long, unramified den- drites that extend
into the neuropil surrounding the VMH (Szentagothai et al., 1968;
Millhouse, 1973b, 1979). VMH dendrites also possess spines
(Szentagothai et al., 1968; Millhouse, 1973b, 1979), small protrusions
that form specialized sites of synaptic contact (Harris and Kater,
1994). Recent Golgi impregnation studies have suggested that spine
density is plastic in a manner that correlates with reproductive
behavior. In particular, the density of dendritic spines on VMH neurons
fluctuates during the estrous cycle, with an increased density
occurring on proestrus compared with diestrus (Frankfurt et al., 1990).
In ovariectomized rats, VMH spine density increased twofold after
estradiol treatment (Frankfurt et al., 1990). Thus, modulation of spine
density in the VMH may be one component of the neural plasticity
induced by estrogen to cause the cyclic changes in reproductive behavior.
Unfortunately, Golgi analysis of VMH neurons has not revealed a
topographical neuronal organization or morphologically distinct cell
types, as has been described in structures like the cerebellum, hippocampus, and neocortex. Consequently, it has not been possible to
make functional inferences about VMH neurons based on their relative
location or neuronal morphology. Furthermore, the Golgi technique does
not allow the concomitant assessment of neurochemical features, such as
the expression of receptors and neuropeptides. To better define the
microcircuitry of the VMH in the present study, Lucifer yellow cell
filling was performed. This technique provided excellent morphological
detail. In addition, this approach was compatible with subsequent
immunostaining for the estrogen receptor. The present results suggest
that the effects of estrogen on spine density are regionally specific
and dendrite specific and are not mediated by a direct action of estrogen.
These results were presented in preliminary form at the Society for
Behavioral Neuroendocrinology, 1999 (Charlottesville, VA).
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MATERIALS AND METHODS |
Animals and hormone treatment. Adult female Sprague
Dawley rats were housed in plastic tubs with standard bedding and with food and water continuously available. The temperature of the colony
was maintained at 22°C with a 12 hr reverse light/dark cycle. Animals
were allowed at least 1 week to acclimate to the colony, then
ovariectomies were performed during anesthesia using aseptic surgical
procedures. After a recovery period of at least 1 week, hormone
treatments began, consisting of subcutaneous injections of vehicle
(n = 4 animals; sesame oil) or estradiol benzoate (EB) (n = 5 animals; 10 µg in 100 µl sesame oil) for 2 consecutive days. Body weight during and after hormone treatment, as
well as uterine size at the time of perfusion, were monitored to
validate the effectiveness of the EB injections. All EB injections were considered effective, based on decreased body weight and increased uterine size compared with vehicle-treated controls.
Perfusion and cell filling. Animals were processed in sets
of three. All sets of animals were processed in close succession, and
each set included both treatment groups. Thus, the histological manipulations for all animals were comparable.
Forty-eight hours after the second hormone injection, animals were
anesthetized with ketamine/xylazine (20 and 80 mg/kg, respectively) and
perfused transcardially with 100 ml saline followed by 200 ml 4%
paraformaldehyde (purified prill; Electron Microscopy Sciences, Fort
Washington, PA). The brains were isolated, and the diencephalon blocks
were post-fixed in paraformaldehyde for 3 hr at 4°C. Coronal sections
were cut on a vibratome at a thickness of 150 µm in 0.1 M
phosphate buffer. The sections were stored in 0.1 M
phosphate buffer at 4°C until cell filling, which occurred within
1-3 d after fixation.
For intracellular injections, sections were placed in 0.1 M
phosphate buffer on a modified stage of a Leica fluorescent microscope. The sections were viewed using differential interference contrast (DIC) optics, and the stage was positioned so that the VMH could be visualized under a 10× long working distance objective.
Intracellular electrodes were constructed from pulled borosilicate
glass capillary tubes (1.0 mm outer diameter/0.58 mm inner diameter;
World Precision Instruments, Sarasota, FL), with the tips broken to a
diameter of ~1 µm. These micropipettes then were backfilled with
4% Lucifer yellow (dilithium salt; Sigma, St. Louis, MO) dissolved in
distilled water. The electrode was advanced toward the section with a
microdrive mounted on a micromanipulator. Individual cells in the VMH
were visualized at 40× magnification under DIC optics and impaled with the electrode. Impalement occurred without intentional bias for any
particular size or shape of cell. The cell then was injected iontophoretically over a 10 min period using  30 nA current. The infusion of Lucifer yellow into a cell was observed with fluorescence, and proper impalement was confirmed by rapid infusion of Lucifer yellow
into a cell, revealing sharp, well defined borders. An average of two
cells were filled per section, five to six sections per animal, which
produced 10-12 filled cells per rat. For analysis, these sections were
mounted onto glass slides and coverslipped using a mounting media that
consisted of glycerol (80%) and 20 mM sodium carbonate
(20%). Coverslips were sealed with nail polish.
Morphological analysis. Confocal microscopy and analysis of
neuronal morphology were performed blind, and the code was not broken
until the data collection was complete. From the 10-12 filled cells
per animal, a subset from each animal was chosen for confocal
microscopy based on the quality of the fill and likely localization in
the VMH (6-7 cells per animal, total = 55 cells). Cells were
visualized with a confocal laser scanning microscope (Leica TCS 4D
System, Leica, Deerfield, IL) using a 100×, 1.4 NA oil-immersion
objective. A single low-power scan (10×) was made of each filled
neuron, followed by a high-power scan (100×) consisting of 9-120
serial, optical sections (0.3-0.5 µm thickness). The total number of
optical sections taken during the high-power scan depended on the
length of the dendrite and the depth that it traveled through the
section, because more optical sections were needed to scan longer,
deeper extending dendrites. Dendritic spines were counted from
individual optical serial sections using NIH software (Image 1.62) on a
Macintosh G3. A confocal projection of overlaid optical sections was
not used for spine counting because such images obscured a large number
of spines above or below the dendrite. In contrast, by viewing the
dendrite through a succession of individual optical serial sections, a
spine obscured by the dendrite in one optical section became apparent
in a successive section(s). However, because no correction was made for
spines situated in planes hidden by the dendrite, spine density
measurements made by this method potentially underestimate the density
of dendritic spines.
The number of spines on each dendritic segment was counted three times,
and the mean was used in the final analysis. To verify experimenter
reliability, spine counts were repeated on one-third of the animals on
two separate occasions, with previous spine counts concealed from the
experimenter. Initial spine counts were highly correlated with the two
subsequent counts (p < 0.0001, r = 0.99).
Other measurements included soma size, cell shape, dendritic length,
and number of primary dendrites and branch points. To establish the
location of a neuron within the VMH, the rostrocaudal position was
determined from landmarks, including the optic tracts and median
eminence. The mediolateral and dorsoventral positions were plotted
using the distance of the cell to the ventral surface of the brain and
to the proximal border of the third ventricle. These coordinates then
were matched against VMH coordinates according to Paxinos and Watson
(1986). A cell was considered to be within the ventrolateral
subdivision of the VMH if it was within the subdivision borders
indicated by Paxinos and Watson (see Fig. 1). Alternatively, in atlas
plates that did not indicate a clear division between the ventrolateral
and dorsal subdivisions, a cell was considered to be within the
ventrolateral subdivision if it was located in the ventral half of the VMH.
Immunocytochemistry. After morphological analysis, sections
were carefully removed from the glass slides for immunostaining procedures. After several washes in TBS, pH 7.4, sections were incubated in estrogen receptor- antisera (1:200, 1D5; Zymed, South
San Francisco, CA) in TBS with 0.2% Triton X-100 and 3% normal donkey
serum (Jackson ImmunoResearch, West Grove, PA) for 1 hr at room
temperature, and then for 72 hr at 4°C. After several washes,
sections were incubated in biotinylated donkey anti-mouse antisera
(1:1000, Jackson ImmunoResearch) in TBS with 0.2% Triton X-100 and 3%
normal donkey serum (Jackson ImmunoResearch) for 4 hr at room
temperature. After several washes, sections were incubated in
cy5-conjugated streptavidin (1:1000, Jackson ImmunoResearch) in TBS
with 0.2% Triton X-100, overnight at 4°C. After final washing, sections were mounted on slides and coverslipped with VectaShield (Vector Laboratories, Burlingame, CA), and coverslips were sealed with
nail polish. Cells were imaged with a confocal laser scanning microscope (Leica TCS 4D System, Leica, Deerfield, IL) using a 40×,
1.25 NA oil-immersion objective.
Statistical analysis. Cells included in the final analysis
had to meet two criteria. First, the soma had to be located within the
VMH, as described above. Fifty-four of the 55 analyzed filled cells met
this criterion. Second, the average spine density for each cell had to
pass the extreme studentized deviate test for outliers.
Fifty-three of the remaining 54 cells met this criteria. For each
dendrite, spine density was calculated as the average number of spines
per millimeter of dendrite. For each neuron, total spine density was
calculated by averaging the spine density for all dendrites. The spine
density for each animal, then, was calculated as an average of the
total spine density for all its filled neurons. Statistical comparisons
were performed by Student's t test (two groups) or ANOVA
(multiple groups). When appropriate, post hoc analysis was
performed using the least significant difference (LSD) test. Circular
statistics were used for comparisons of spatial orientation analysis,
namely the Rayleigh and the  2 test
(Batschelet, 1981). Significance was set at p < 0.05.
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RESULTS |
A total of 53 Lucifer yellow-filled neurons in the VMH were
included in the final analysis (five to six cells per animal, nine
animals). Most of these neurons resided in the ventrolateral VMH
(vlVMH, 60%, n = 31 cells), with the remaining neurons
in the dorsal subdivision (40%, n = 22 cells) (Fig.
1). The morphological features of the
Lucifer yellow-filled neurons were similar to those previously observed
in other studies using Golgi impregnation (Table
1) (Szentagothai et al., 1968; Millhouse,
1973b, 1979). Most neurons were spherical or ovoid with long,
relatively straight, sparsely branched, spinous dendrites. On average,
there were two primary dendrites per neuron, with approximately one
branch point in each dendritic tree. Although most dendrites were
straight, some were twisted. In most cases the end of the dendrite was
tapered, but in other cases it was rounded. Axons were occasionally
observed and appeared as thin, beaded processes. Confocal microscopic
imaging of Lucifer yellow-filled neurons provided excellent
visualization of dendritic spines.
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Figure 1.
Composite drawings of the locations of Lucifer
yellow-filled cells within the VMH from vehicle-treated ( ) and
EB-treated ( ) rats. Cell location was estimated by matching the
measured distance of the cell from the ventral surface of the brain and
from the third ventricle with coordinates from Paxinos and Watson
(1986). Numbers to the right indicate
each section's coordinates posterior from Bregma. Arc,
Arcuate; ME, median eminence; VMH,
ventromedial hypothalamic nucleus; dmVMH, dorsomedial
subdivision of the VMH; vlVMH, ventrolateral subdivision
of the VMH. Drawings are modified tracings based on Paxinos and Watson
(1986).
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Estrogen treatment increased the average density of dendritic spines in
the vlVMH per neuron per animal by 48% (p < 0.05, Student's t test) (Fig.
2). This effect was region-specific,
because no effect of estrogen was observed in the dorsal VMH. Examples of dendritic spines for each group are shown in Figure
3.
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Figure 2.
Comparison of dendritic spine density in the
ventrolateral VMH and dorsal VMH from vehicle-treated
(n = 4) and EB-treated (n = 5)
rats (mean ± SEM). EB treatment significantly increases spine density
in the ventrolateral but not the dorsal VMH. Spines were counted from
confocal movie images, and length was measured using NIH Image 1.62. Spine density values are expressed in number of spines per millimeter
dendrite per cell per rat. Ventrolateral VMH: two to five cells/animal,
4-17 dendrites/animal. Dorsal VMH: one to four cells/animal, 2-13
dendrites/animal. Asterisk indicates
p < 0.05, Student's t test.
Veh, Vehicle; EB, estradiol benzoate;
vl, ventrolateral.
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Figure 3.
Digital micrographs from a confocal projection of
representative dendrite segments from vehicle- and EB-treated rats.
Individual serial optical sections were overlaid to create this figure.
Actual spine counts were made from individual optical sections rather
than an overlaid image as seen here. Arrows point to
examples of apparent dendritic spines in the overlaid image. Certain
spines appeared with more clarity in individual optical sections.
Confocal images were converted to gray scale and inverted to a negative
image using Adobe Photoshop 4.0. Scale bar, 10 µm.
Veh, Vehicle; EB, estradiol
benzoate.
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Subsequent analysis focused on the dendritic arbor of vlVMH neurons.
Dendrites of vlVMH neurons were segregated into mutually exclusive
categories that differed significantly in their basic characteristics
and in their response to estrogen. Dendrites were classified as either
long primary (longest primary dendrite), short (non-longest) primary,
or secondary. By definition every neuron had only one "long"
primary dendrite. vlVMH neurons had an average of 1.0 ± 0.2 short
dendrites per neuron (mean ± SEM). On average, the long primary
dendrites were fourfold longer than the dendrites in the remaining
categories (Fig. 4). Estrogen treatment, however, did not affect the length of any type of dendrite. Thus, the
estrogen-induced increase in spine density, described above, could not
be explained by a decrease in dendrite length. The long and short
primary dendrites also differed in their overall spatial orientations
(p < 0.01, 2
test), which were classified as either dorsolateral, ventrolateral, ventromedial, or dorsomedial. The long primary dendrites were significantly oriented in the ventrolateral direction (vector length = 0.4909, p < 0.01, Rayleigh test),
whereas short primary dendrites showed no directional preference (Fig.
5). Thus, this classification of primary
dendrites can be justified based on both length and spatial orientation
differences.
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Figure 4.
Comparison of the lengths of the long primary,
short primary, and secondary ventrolateral VMH dendrites (mean ± SEM,
n = number of dendrites). Estrogen treatment did
not affect the length of any dendrite type. Dendrite length depended on
dendrite class
(F(2,80) = 22.26, p < 0.01, two-way ANOVA). The mean length
of the long primary dendrites was significantly different from those of
the other dendrite types. Lengths were measured using NIH Image 1.62. Asterisk indicates p < 0.01 on
post hoc analysis (LSD). veh, Vehicle;
EB, estradiol benzoate.
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Figure 5.
Spatial orientation of long and short primary
dendrites of the ventrolateral VMH. Dendrites were classified as
projecting in the ventromedial (vm), ventrolateral
(vl), dorsolateral (dl), or
dorsomedial (dm) direction. Long primary dendrites had a
significant preference for the ventrolateral direction
(2 test and Rayleigh test, p < 0.05), whereas short primary dendrites were randomly oriented.
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Subsequent analysis addressed whether estrogen treatment differentially
regulated spine density on the long and short primary dendrites of
vlVMH neurons. The effect of estrogen on spine density differed
depending on dendrite category
(F(1,61) = 4.20, p < 0.05, two-way ANOVA). Estrogen increased spine
density by approximately 2.5-fold on the short primary dendrites
(p < 0.05, LSD) without affecting spine density
on the long primary dendrites (Fig. 6). Estrogen treatment did not alter spine density on the secondary dendrites.
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Figure 6.
Comparison of spine densities of the long primary,
short primary, and secondary dendrites of ventrolateral VMH cells from
vehicle- and estrogen-treated rats (mean ± SEM, n = number of dendrites). The effect of estrogen depended on dendrite
class (F(1,61) = 4.20, p < 0.05, two-way ANOVA). Estrogen increased
spine density in the short primary dendrites without affecting spine
density on the long primary dendrites. Asterisks
indicates p < 0.05 on post hoc
analysis (LSD). Estrogen treatment did not alter spine density on
secondary dendrites. veh, Vehicle; EB,
estradiol benzoate.
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Detection of spine induction on the short, but not the long, primary
dendrites would occur if estrogen treatment has differential effects on
the two types of dendrites. Alternatively, estrogen might induce spines
close to the soma on both types of dendrites. If so, the unaffected
distal regions of the long primary dendrites might obscure an effect on
these dendrites. To examine this possibility, spines were recounted on
50 µm segments of the primary dendrites. Both proximal (0-50 µm
from the soma) and distal (100-150 µm and 150-200 µm from the
soma) dendritic segments were analyzed. Within the first 50 µm of
dendrite, the effect of estrogen on spine density differed between long
and short dendrites
(F(1,61) = 5.44, p < 0.05, two-way ANOVA). Estrogen increased spine
density on the first 50 µm of the short primary dendrites
(p < 0.05, LSD), but did not affect spine
density on the first 50 µm of the long primary dendrites (Fig.
7).
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Figure 7.
Comparison of spine density on 50 µm dendritic
segments of the long and short primary dendrites of ventrolateral VMH
neurons from vehicle- and estrogen-treated rats (mean ± SEM, n = number of dendrites).
Within the first 50 µm of dendrite, the effect of estrogen on spine
density differed between long and short dendrites
(F(1,61) = 5.44, p < 0.05, two-way ANOVA). Estrogen increased spine
density on the first 50 µm of the short primary dendrites, but did
not affect spine density on the first 50 µm of the long primary
dendrites. On the long primary dendrites, the effect of estrogen on
spine density did not depend on proximity to soma nor did proximity to
soma itself affect spine density. However, overall, estrogen treatment
decreased spine density
(F(1,67) = 10.66, p < 0.01, two-way ANOVA). This decrease was
significant on dendritic segments 100-150 µm from the soma.
Asterisks indicate p < 0.05 on
post hoc analysis (LSD). veh, Vehicle;
EB, estradiol benzoate.
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By comparing proximal and distal segments, spine density on the long
primary dendrites then was assessed for a possible interaction between
estrogen treatment and distance from the soma. The corresponding analysis on the distal segments of short dendrites could not be performed because very few dendrites in this category extended beyond
100 µm. Proximity to soma itself did not affect spine density significantly on the long primary dendrites. In addition, the effect of
estrogen treatment did not depend on proximity to soma. However,
estrogen treatment did decrease spine density on the long primary
dendrites (F(1,67) = 10.66, p < 0.01, two-way ANOVA). This decrease was
significant on the dendritic segment 100-150 µm from the soma
(p < 0.05, LSD) (Fig. 7).
Because vlVMH is abundant in estrogen receptor-containing neurons
(Pfaff and Keiner, 1973; Simerly et al., 1990; DonCarlos et al., 1991;
Shugrue et al., 1992), it is reasonable to propose that in this brain
region estrogen-induced spines may occur on neurons that express
estrogen receptor. If so, spine induction may be mediated by a direct,
genomic effect of estrogen within the cell. Alternatively, if spine
induction occurs in neurons that do not express estrogen receptor, such
a direct mechanism would seem unlikely. To elucidate the mechanisms of
estrogen-induced spines in the VMH, sections with Lucifer yellow-filled
cells in the vlVMH were immunostained with an estrogen receptor-
antibody. Previous studies have demonstrated that immunostaining is
compatible with Lucifer yellow cell filling and that Lucifer yellow
does not interfere with the immunoreactivity of other antigens (Kawata et al., 1983; Pilowksy et al., 1991). Consistent with earlier studies
(Pfaff and Keiner, 1973; Simerly et al., 1990; Shugrue et al., 1992),
abundant nuclear staining for the estrogen receptor was visible in the
area around the ventrolateral border of the vlVMH. Some of the Lucifer
yellow-filled cells were in close proximity to estrogen
receptor-containing cells. However, the majority of Lucifer
yellow-filled cells in the vlVMH were somewhat medial to the estrogen
receptor cluster. All 31 of the Lucifer yellow-filled cells located in
the vlVMH were clearly visible after immunostaining and could be easily
distinguished from the immunostained cells. Nevertheless, in both
groups, none of the Lucifer yellow-filled cells were co-labeled for
nuclear estrogen receptor- (Fig. 8). The Lucifer yellow-filled cells were predominantly located in the
medial portion of the vlVMH, whereas the estrogen receptor cluster was
visible in the area near the ventrolateral border of the vlVMH. Thus,
based on their location, it is not unusual that none of the Lucifer
yellow-filled cells were labeled for estrogen receptor-. The lack of
estrogen receptor- in the Lucifer yellow-filled cells suggests that
spine induction within these neurons does not occur through a direct,
genomic mechanism. This interpretation allows for a genomic effect on
the nearby estrogen receptor--containing neurons, which may
transynaptically induce spines on cells that do not possess estrogen
receptor-. Such a mechanism has been proposed in the hippocampus
(Murphy et al., 1998). Although we document spine density changes on
neurons lacking estrogen receptor-, it remains to be determined
whether such changes occur on estrogen receptor--positive
neurons.
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Figure 8.
Digital micrographs of representative
Lucifer yellow-filled cells and estrogen receptor-containing neurons in
the ventrolateral VMH. A, Estrogen receptor-
immunoreactive neurons labeled with cy5. B, Composite
image of cy5-labeled estrogen receptor- immunoreactive neurons and
Lucifer yellow-filled cell. Only a portion of the dendritic arbor of
the Lucifer yellow-filled cell is shown. Confocal images were converted
to gray scale and inverted to a negative image using Adobe Photoshop
4.0. White arrowheads indicate estrogen
receptor--immunoreactive neurons. Black arrowhead
indicates (B) a Lucifer yellow-filled neuron or
(A) the location of a Lucifer yellow-filled
neuron. Scale bar, 50 µm.
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DISCUSSION |
The goal of this study was to elucidate the neural elements of the
microcircuitry within the VMH that exhibit estrogen-induced neural
plasticity. Previous work has shown that estrogen increases dendritic
spine density in the VMH (Frankfurt et al., 1990). Our results go
beyond these original observations in three important ways. First,
within the VMH the induction of spines is subdivision specific. Second,
although the dendritic arbors of VMH neurons are simple, anatomical
evidence exists for dendrite specialization. This is supported by the
finding that estrogen specifically induces spines in short primary
vlVMH dendrites. Third, spine induction by estrogen occurs on neurons
that, based on immunostaining, do not express nuclear estrogen
receptor-. Each of these findings is discussed in turn.
Initial studies used Golgi impregnation to define the morphology of VMH
neurons (Szentagothai et al., 1968; Millhouse, 1973b, 1979). Our
results with Lucifer yellow-filled cells revealed morphological features similar to those described, particularly with regard to cell
size, dendrite number, and dendrite length (Szentagothai et al., 1968,
Frankfurt et al., 1990; Millhouse, 1973b, 1979). Overall,
somewhat higher spine density values were obtained with Lucifer yellow
cell filling (100-300 spines/mm) than have been detected with Golgi
(50-200 spines/mm) (Frankfurt et al., 1990; Frankfurt and McEwen,
1991a). A likely explanation for this discrepancy is that
Golgi-impregnated cells were analyzed using camera lucida drawings
under light microscopy, whereas the Lucifer yellow-filled cells were
visualized using confocal microscopy, which allows the detection of
spines that would otherwise be obscured by the dendrite. Overall, these
two techniques provide comparable morphological results.
Our observation that estrogen-induced spines are localized to the vlVMH
is noteworthy, given the evidence that this subdivision is involved in
female sexual behavior. Sexual behavior selectively induces immediate
early gene expression in the vlVMH but not the dorsal VMH (Flanagan et
al., 1993; Pfaus et al., 1993; Tetel et al., 1993; Polston and Erskine,
1995). Additionally, estrogen receptor-containing neurons are found
specifically in the vlVMH (Pfaff and Keiner, 1973; Simerly et al.,
1990; DonCarlos et al., 1991). Moreover, the descending projections of
the ventrolateral but not the dorsal VMH target the ventrolateral
periaqueductal gray (Canteras et al., 1994), the subdivision thought to
be involved in the lordosis posture (Lonstein and Stern, 1998; Daniels
et al., 1999). Although the regional specificity of estrogen-induced spines in the VMH implicates them in sexual behavior, it remains to be
established whether these spines represent legitimate synapses. Another
question about the function of the induced spines is whether they
contribute to the priming actions of estrogen for sexual behavior or to
the execution of this behavior.
In addition to regional specificity, we also found that the effects of
estrogen are spatially organized within the dendritic tree. The simple
arborization of VMH dendrites has been described previously
(Szentagothai et al., 1968; Millhouse, 1973b, 1979). The present study
found differences in the length and direction of the primary dendrites
that suggested functional differences. For instance, the short primary
dendrites may receive input from local vlVMH neurons. In contrast,
long, ventrolaterally oriented dendrites may receive input from the
neuropil, which is rich with extrinsic afferents and runs along the
lateral edge of the VMH (Millhouse, 1973a). Further evidence for
differential innervation of these two dendrite types was provided by
the differential effects of estrogen on their spine density. Spine
density is differentially regulated based on dendrite-specific afferent
input. For instance, in hippocampal cultures, afferent input to
specific dendrites alters spine density on those dendrites without
generalized effects on the remaining dendrites (Kossel et al., 1997).
Thus, short primary dendrites in the vlVMH may receive
estrogen-modulated afferent input that is unavailable to the long
primary dendrites.
The dendrite specificity of estrogen-induced spines in the VMH may not
have been detected in previous studies that analyzed only a single
primary dendrite, specifically, the primary dendrite with the
"greatest number of spines" (Frankfurt et al., 1990) or the
"second longest primary dendrite" (Segarra and McEwen, 1991).
Because results of the former study are similar to those of the present
study, it is possible that the sample consisted mainly of short
dendrites. The second longest primary dendrite of the latter study
would be a subset of our short primary dendrites. Thus, the present
results replicate previous findings, but our more complete analysis of
the dendritic tree has revealed that the effect of estrogen is
selective within the dendritic arbor.
In addition to spine induction, we have detected a decrease in spine
density on the distal portions of the long primary vlVMH dendrites.
This result suggests that differential afferent input occurs along the
length of long dendrites such that estrogen selectively alters the
strength of inputs within specific segments of the long dendrites.
Consequently, the effect of estrogen differs not only between dendrite
categories (long vs short), but also within specific segments of the
long dendrites. The concomitant spine density increase in short
dendrites and decrease in long dendrites may explain why estrogen
treatment did not seem to alter the number of axospinous synapses in
the estrogen-treated rat vlVMH in previous electron microscopy studies
(Nishizuka and Pfaff, 1989; Frankfurt and McEwen, 1991b). In
particular, averaging of both dendrite types may have concealed the
effect of estrogen on these synapses.
To explore the cellular mechanisms of estrogen-induced spines, we
immunostained Lucifer yellow-filled vlVMH cells for estrogen receptor-. None of the cells in either treatment group expressed nuclear estrogen receptor. This suggests that estrogen did not act
through direct, genomic mechanisms within these neurons to increase
spine density. Instead, estrogen may act indirectly, altering activity
afferent to the filled neurons. Many studies in the telencephalon have
shown that the induction and maintenance of dendritic spines depends on
afferent input (Annis et al., 1994; Bundman et al., 1994;
Kossel et al., 1997; McKinney et al., 1999), particularly through
glutamate receptors (Goldowitz et al., 1979; Kossel et al., 1997;
McKinney et al., 1999). In fact, estrogen induces spines through a
glutamate receptor-dependent mechanism in hippocampal CA1 neurons
(Woolley and McEwen, 1994; Murphy and Segal, 1996). The role of
glutamate in estrogen-induced vlVMH spines is unclear. However, several
lines of circumstantial evidence support this mechanism. First, NMDA
and AMPA receptors have been localized to the VMH (Brann and Mahesh,
1994; Meeker et al., 1994; van den Pol et al., 1994, 1995; Mateos et
al., 1998). Second, estrogen increases VMH glutamate levels (Mansky and
Wuttke, 1983; Frankfurt et al., 1984; Luine et al., 1997). Third,
estrogen priming of rat sexual behavior requires glutamatergic receptor
activation (Fleischmann et al., 1991). However, because hypothalamic
spine induction mechanisms have not been well studied, it seems
premature to rule out the contributions of other neurotransmitters
(Frankfurt and McEwen, 1991a).
Regardless of the neurotransmitter system involved, the effect of
estrogen on cells lacking estrogen receptor suggests that estrogen acts
transynaptically, rather than directly, to induce dendritic spines. The
key question, then, is whether the afferents that stimulate spine
formation are extrinsic or intrinsic to the VMH. Previous anatomical
studies have suggested that extrinsic afferents to the vlVMH are found
in the neuropil surrounding the VMH (Millhouse, 1973a; Swanson and
Hartman, 1975). In support of the importance of extrinsic afferents for
estrogen-induced neural plasticity, VMH denervation prevented
estrogen-induced axodendritic synapse formation (Nishizuka and Pfaff,
1989). The length and ventrolateral orientation of the long dendrites
suggest that they are more likely to be innervated by the afferents in the neuropil. It is currently unclear how extrinsic afferent
innervation on long dendrites would lead to increased spines on short
dendrites. Alternatively, estrogen may increase local afferent activity
on the short dendrites, leading to increased spine formation on these same dendrites. This afferent input may be provided by nearby estrogen
receptor-containing vlVMH neurons (Fig.
9). The fact that the spine density
changes occur in neurons lacking estrogen receptor might be interpreted
to suggest that this phenomenon is not related to sexual behavior.
However, the location of our filled cells closely matches the effective
stimulation sites used in a previous study using electrical stimulation
within the VMH to facilitate sexual behavior, i.e., medial to the
cluster of estrogen receptor-containing cells (Pfaff and Sakuma,
1979b).
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|
Figure 9.
Model of possible mechanisms of spine induction on
short primary dendrites in the ventrolateral VMH. The effect of
estrogen on cells lacking estrogen receptor suggests that estrogen acts
transynaptically, rather than directly, to induce dendritic spines. The
afferents that stimulate spine formation may be either extrinsic or
intrinsic to the VMH. Extrinsic afferents are mainly found in the
neuropil surrounding the VMH. Long primary dendrites extending to the
ventrolateral border of the VMH may be innervated by extrinsic
afferents in the neuropil. Intrinsic afferents may innervate short
primary dendrites. We propose that estrogen affects local intrinsic
afferents to increase spine density on short primary dendrites.
Open circles indicate excitatory synapses;
arrows indicate direction of information flow through
the circuit. VMH, Ventromedial nucleus of the
hypothalamus; PAG, periaqueductal gray.
|
|
In conclusion, the effects of estrogen on reproductive behavior appear
to involve various changes in the synaptic organization of the vlVMH,
including dendrite-specific changes in spine density. These results
suggest that estrogen differentially modulates the strength of multiple
afferents to vlVMH neurons. However, the source of these afferents and
the site and mechanism of estrogen action remain as key questions. The
fact that this phenomenon occurs in a brain region critical for female
sexual behavior suggests that the induced spines may be involved in the
priming action of estrogen and/or actual execution of receptive
behaviors. The lack of estrogen receptor immunoreactivity in these
cells suggests that spine induction is mediated by an indirect
mechanism possibly involving afferent stimulation. Future studies are
needed to determine (1) the phenotype and function of the neurons that
exhibit estrogen-induced spines and (2) whether estrogen
receptor--containing neurons also exhibit estrogen-induced dendritic spines.
| |
FOOTNOTES |
Received Aug. 3, 1999; revised Nov. 19, 1999; accepted Nov. 29, 1999.
L.M.F.-C. is supported by National Institutes of Health Grants MH54712,
MH43787, and DK52018. We thank Dr. D. Perkel for generously making his
equipment and expertise available to us. We also thank Dr. R. Balice-Gordon, Dr. A. Christie, S. Benton, and J. Cardin for their
technical advice, and Drs. D. Perkel and R. Balice-Gordon for their
comments on an earlier version of this manuscript.
Correspondence should be addressed to Lyngine H. Calizo, Department of
Neuroscience, University of Pennsylvania, 215 Stemmler Hall, 36th and
Hamilton Walk, Philadelphia, PA 19104-6074.E-mail: lcalizo{at}mail.med.upenn.edu.
| |
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ABSTRACT
INTRODUCTION
