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The Journal of Neuroscience, February 15, 1999, 19(4):1464-1472
Gonadal Steroids Promote Glial Differentiation and Alter Neuronal
Morphology in the Developing Hypothalamus in a Regionally Specific
Manner
Jessica A.
Mong1,
Edmund
Glaser2, and
Margaret M.
McCarthy2
1 Department of Pharmacology and Experimental
Therapeutics and 2 Department of Physiology and Program in
Neuroscience, University of Maryland School of Medicine, Baltimore,
Maryland 21201
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ABSTRACT |
One of the more striking sexual dimorphisms in the adult brain is
the synaptic patterning in some hypothalamic nuclei. In the arcuate
nucleus (ARC) males have twice the number of axosomatic and one-half
the number of axodendritic spine synapses as females. The opposite
pattern is observed in the immediately adjacent ventromedial nucleus
(VMN). In both cases, early exposure to testosterone dictates adult
dimorphism, but the exact timing, mechanism, and site of steroid action
remain unknown. Astrocytes also exhibit sexual dimorphisms, and their
role in mediating neuronal morphology is becoming increasingly evident.
Using Golgi-Cox impregnation to examine neuronal morphology and glial
fibrillary acidic protein immunoreactivity (GFAP-IR) to
characterize astrocytic morphology, we compared structural differences
in dendrites and astrocytes from the ARC and VMN in postnatal day 2 rat
pups from four hormonally different groups. Consistent with previous
observations, testosterone exposure induced a rapid and dramatic
stellation response in ARC astrocytes. Coincident with this change in
astrocytic morphology was a 37% reduction in the density of dendritic
spines on ARC neurons. In contrast, astrocytes in the VMN were poorly
differentiated and did not respond to testosterone exposure, nor were
there any changes in neuronal dendrite spine density. However, VMN
neurons exposed to testosterone had almost double the number of
branches compared with that in controls. These data suggest that the
degree of maturation and the differentiation of hypothalamic astrocytes in vivo are correlated with the ability of neurons to
sprout branches or spines in response to steroid hormones and may
underlie regionally specific differences in synaptic patterning.
Key words:
sexual differentiation; arcuate; ventromedial nucleus; estrogen; spine density; developmental synaptic plasticity; GFAP; Golgi-Cox
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INTRODUCTION |
The perinatal developmental period
is characterized by dramatic sex differences in circulating gonadal
steroids in a variety of mammalian species including humans (Gerall et
al., 1992 ). On embryonic day 18, the testes of developing male rats
secrete adult levels of testosterone that gradually decline until a
second peak of secretion on the day of birth. In females, exposure to
gonadal steroids remains uniform and low (Weisz and Ward, 1980). This dimorphic exposure to steroids has profound and permanent effects on
the developing brain. In the rodent, it is well established that
estrogens, the products of aromatizable androgens, mediate many aspects
of sexual differentiation of the male brain during a restricted
development period from embryonic day 18 to postnatal day 6-10
(Maclusky and Naftolin, 1981; Arnold and Gorski, 1984; Gerall et al.,
1992). Exposure to estrogens during this critical period results in a
"masculinized" neural pattern in the adult rat, whereas a lack of
such exposure yields a "feminized" brain. A major consequence of
this dissimilar steroid exposure is the sexually dimorphic synaptic
patterning in the arcuate nucleus (ARC) and ventromedial nucleus (VMN),
two hypothalamic nuclei that play critical roles in the control of
gonadotropin secretions from the pituitary (Ojeda and Urbanski, 1994)
and stereotypic female sex behavior (Pfaff et al., 1994), respectively.
Quantitative electron microscopy studies have shown that in the ARC,
females have approximately twice the number of axodendritic spine
synapses as males and this patterning is sensitive to hormonal
manipulation during the critical period for brain sexual
differentiation (Matsumoto and Arai, 1980). The opposite pattern is
observed in the ventrolateral VMN where adult males have twice as many
dendritic spine synapses as females (Matsumoto and Arai, 1986).
The cellular and molecular mechanisms mediating steroid-induced changes
in synaptic patterning are unknown. Recently, several lines of evidence
have focused on neuronal-glial interaction as a possible mechanism for
mediating synaptic connectivity. We have demonstrated previously that
astrocytes in the ARC of neonatal rats exhibit a sexually dimorphic
morphology as early as postnatal day 1 (PN1). In gonadally intact
males, the ARC has a greater frequency of fully differentiated
astrocytes, as evidenced by their complex morphology. As with synaptic
patterning, this sex difference in astrocyte morphology is sensitive to
hormonal manipulation. Castrate males and females treated with
testosterone propionate demonstrated dramatic increases in astrocyte
differentiation, whereas removal of testosterone by gonadectomy
resulted in a lack of differentiation within 24 hr (Mong et al., 1996).
These data suggest that astrocytes may participate in synaptic
patterning by guiding or blocking the path of neurites. As a first step
in testing the hypothesis that steroid-mediated changes in astrocytic morphology determine sexually dimorphic synaptic patterning, we have
observed coordinated changes in the dendritic morphology of
Golgi-Cox-impregnated neurons and in the morphology of astrocytes expressing glial fibrillary acidic protein (GFAP) in the ARC and VMN of
PN2 rats that had been hormonally manipulated on the day of
birth and on PN1. We examined regionally specific astrocyte maturation
further by Western blot analysis of GFAP levels.
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MATERIALS AND METHODS |
Animals
Female Sprague Dawley rats (Charles River Laboratories,
Wilmington, MA) were mated in our animal facility, and pregnancy
was confirmed by the presence of sperm in the vaginal smear. Pregnant females were isolated and allowed to deliver normally. Animals were
maintained on a reversed 12:12 hr light/dark cycle (lights out at 10:00
A.M.) and were given food and water ad libitum. Cages were checked regularly for the presence of pups to determine the day of
birth (PN0). Only litters that were found in the morning were used in
subsequent experiments. Castrations were performed on PN0 under cold
anesthesia, and gonadally intact male and female pups underwent sham
operations. All pups were toe-clipped for group identification. After
being warmed to 37°C, pups were returned to their mothers. All the
pups received two subcutaneous injections 24 hr apart of either 100 µg of testosterone propionate (TP) in a 0.1 cc volume of sesame oil
or 0.1 cc of sesame oil alone. Brains were collected on PN2, ~24 hr
after the last hormonal treatment. The treatment groups were as
follows: (1) castrate males injected with oil vehicle
(n = 6); (2) gonadally intact males injected with oil
vehicle (n = 8); (3) gonadally intact females injected with TP (n = 6); and (4) gonadally intact females
injected with oil vehicle (n = 7).
Golgi-Cox impregnation
Brains were embedded according to the methods of Glaser and Van
der Loos (1981) with some modifications. Briefly, the neonates were
overdosed with pentobarbital before being transcardially perfused with
0.9% saline, and the brains were placed in 30 ml of Golgi-Cox solution
(1:1 solution of 5% K2Cr207 and
5% HgCl2, which is then added to 5%
K2CrO4 in a 4:10 ratio). The brains were placed
in fresh Golgi-Cox solution after 48 hr and remained in it for 20 d. After impregnation, the brains were placed in 30% sucrose in
dH2O for 3 d, then cut on a vibratome at 100 µm, and
mounted on 2% gelatin-subbed glass slides as described previously (Forgie et al., 1996; Gibb and Kolb, 1998). The sections were developed
and counterstained with methylene blue according to the procedure
outlined in Glaser and Van der Loos (1981).
Immunocytochemistry: GFAP immunoreactivity
Animals used for GFAP immunoreactivity (GFAP-IR) received
the same hormonal treatment (n = 4 for each group) as
the Golgi-Cox-impregnated brains and were killed by decapitation ~24
hr after the last hormonal treatment (PN2). The brains were removed and
submersion fixed in 0.1% acrolein and 4% paraformaldehyde, followed
by incubation in 30% sucrose in PBS for cyroprotection. After
fixation, the brains were frozen on dry ice and stored at  80°C
until processed for immunocytochemistry.
Brains were sectioned into 50-µm-thick sections along the sagittal
plane using a cryostat. The sections were placed into wells containing
0.1 M PBS, washed twice in PBS, and incubated with 1% H2O2 in 100% methanol for 15 min. They
were then washed three times in PBS, followed by a 1 hr incubation in
10% normal goat serum and 0.3% Triton X-100 in PBS. After blocking, a
mouse monoclonal GFAP primary antibody (Boehringer Mannheim, Mannheim,
Germany) was applied to the sections at a 1:10,000 dilution in 10%
normal goat serum and 0.3% Triton X-100 in PBS, and the sections were incubated overnight at 4°C. After primary incubation, the sections were washed three times in PBS and incubated in biotinylated secondary antibody (goat anti-mouse; Vector Laboratories, Burlingame, CA) for 1 hr, followed by three washes in PBS. Finally, the samples were
incubated with an avidin-biotin horseradish-peroxidase complex (Vectastain ABC, Elite Kit; Vector Laboratories) for 1 hr at room temperature, washed twice with PBS, and visualized with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Polysciences, Warrington, PA)
and 0.005% H2O2. After visualization, the
sections were mounted serially on 2% gelatin-coated glass slides.
Morphological analysis
Dendrites. Golgi-Cox-impregnated dendrites were
analyzed by computerized reconstruction (Glaser and Van der Loos,
1981). Slides were numerically coded, and the reader was blind to the
experimental group. Anatomically matched sections (two to three) were
analyzed from each animal for all groups. These sections corresponded
to embryonic day 22 (E22) coronal plate 15 in the prenatal rat
atlas of Altman and Bayer (1995) and contained both the ventrolateral VMN and a rostral portion of the ARC (Fig.
1A). Neurons that
appeared well impregnated were marked for analysis. A 20× Nikon
objective was used for this selection procedure so that the observer
would not be biased by the appearance of spines on the dendrites. A neuron was considered well impregnated if its cell body was filled with
the black precipitate and the dendrites were filled to their ends.
Because axons were only occasionally filled, and rarely to their ends,
their impregnation was not a criterion for determining well-impregnated
neurons. Ten to twenty dendrites in both the VMN and ARC of each animal
were randomly selected and analyzed for all groups. Well-impregnated
dendrites were then reconstructed through the different focal planes of
the section under a 100× oil-immersion objective using Neurolucida, an
image-combining computer microscopy program (MicroBrightField,
Colchester, VT). In the dendritic reconstruction, spines, branch
points, and total dendritic length were measured. A spine was defined
as any protrusion under 5 µm, and any protrusion over 5 µm in
length was considered to be a branch point. Branch points represented
the number of times a primary dendrite divided into subsequent
branches. Total dendritic length was measured in micrometers and
includes all branches on the primary dendrite. Spine density was
calculated by dividing the total number of spines per dendrite by the
total length of the dendrite. Spine densities and total dendritic
length were reported as the means for each treatment group.
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Figure 1.
Anatomical regions analyzed for neuronal and
astrocytic morphology. A, Representative
photomicrographs (2×) of a coronal section from a
Golgi-Cox-impregnated PN2 rat brain (top) and camera
lucida drawings of the ARC and VMN in the
coronal plane (bottom). Impregnated neurons were
reconstructed with computerized microscopy from the ventrolateral
portion of the VMN (vl-VMN) and
the ARC. B, Representative
photomicrographs (2×) of GFAP-IR in the sagittal plane of a PN2 rat
(top) and camera lucida drawings of the
ARC and VMN in the sagittal plane of a
neonatal rat brain (bottom). The astrocyte process
length and the degree of differentiation were analyzed in the
ARC of sagittal sections to avoid the appearance of
GFAP-IR tanicytes that are specialized glia present in the
ARC and median eminence. Scale bar, 500 µm.
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GFAP-IR astrocytes. In sagittal sections, the arcuate
nucleus appears as a distinct cell population in the region just
lateral to the third ventricle. It was necessary to section the tissue for GFAP-IR in the sagittal plane because in coronal sections differentiated GFAP-IR astrocytes are obscured by GFAP-IR
tanicytes, specialized glial cells found in the ARC and median
eminence. The VMN also appears in the same sections as the ARC. It is
an oblong-shaped nucleus just anterior to the ARC (Fig.
1B). To analyze cells from the same anatomical region
of the ARC and VMN for each animal, we selected the first four sections
starting ~100 µm lateral to the third ventricle. These sections
corresponded to E22 sagittal plate 2 in the prenatal atlas of Altman
and Bayer (1995) (Fig. 1B). Slides were numerically
coded, and the investigator conducting the analysis was blind to the
experimental group. GFAP-IR cells that could be identified as one
individual cell were selected for analysis. The National Institutes of
Health IMAGE software program was used to measure the process length of
GFAP-immunoreactive cells in the ARC of anatomically matched sections
across the four groups. The process length of GFAP-IR cells in the VMN
was not quantifiable (see Results). By the use of the line selection
tool, the path of a GFAP-IR process was traced from the center of the cell soma to its end. Using the measurement option, we measured the
process length in micrometers. Three to five processes were measured
per cell, and their mean process length per cell was determined. Seven
to ten cells per section were selected for measurement, and four
sections per brain from four animals per group were analyzed.
For further analysis, all the GFAP-IR glial cells measured for process
length in the ARC of the four treatment groups were then categorized
into four arbitrary classes based on their morphological shape and
degree of branching. This classification scheme was designed after
observing distinct differences in glia morphology between the treatment
groups (Mong et al., 1996). Class I cells were characterized as having
only primary processes that were short and thick. Class II and class
III cells were more bipolar in shape with extended processes. Class II
cells possessed primary and some secondary processes for a total of
5-15, whereas class III cells had more secondary processes, usually
15-30. Finally, class IV cells were characterized by their long thin
extended processes that possessed a high degree of branching (secondary and tertiary processes). In addition, the class IV cells were stellate
in contrast to the bipolar morphology of classes I-III. Representative
photomicrographs of the GFAP-IR astrocytes from the different classes
are presented in Figure 2. Only GFAP-IR cells that were distinguishable as individual astrocytes were classified. Approximately 25 GFAP-IR cells per animal and four animals
per group were classified. After classification, the percentage of
astrocytes in each class was determined from the total number of cells
classified for that brain.
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Figure 2.
Representative photomicrographs (40×) of classes
of GFAP-IR cells in the ARC of PN2 rats. To characterize
hormone-mediated changes in astrocyte morphology further, we developed
a classification scheme that divided cells into four arbitrary classes
based on increasing complexity. Class I cells are characterized by only
primary processes that extend from the soma. Class II cells have an
overall bipolar shape, and their processes branch ~5-15 times. Class
III cells are characterized by a more stellate appearance but still
retain some bipolar morphology and have an increased number of branches
(15-30) compared with that in Class II. Class IV cells are
characterized by a fully stellate morphology. Scale bar, 25 µm.
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Western blotting
Unmanipulated male and female pups (n = 7) were
killed by decapitation ~48 hr after birth (PN2). The brains were
removed and frozen on dry ice and stored at 70°C until being
processed for micropunching. Brains were cut in the coronal plane into
300-µm-thick sections on a cryostat. The sections were collected onto
glass microscope slides and immediately frozen. The ARC and
ventrolateral VMN were microdissected according to the micropunch
technique of Palkovits (Palkovits and Brownstein, 1988). The tissue
punches were placed in 50 µl of 10 mM HEPES, pH 7.2, and
sonicated for 10 sec. An aliquot was removed to determine protein
content, and the remaining tissue was denatured with SDS (1%
final concentration; Sigma, St. Louis, MO) and  -mercaptoethanol (1%
final concentration; Sigma) and stored at 70°C until processing
(Laemmli, 1970; Towbin et al., 1979)
Protein (5 µg) from the ARC and VMN was electrophoresed in separate
lanes on an 8-16% precast SDS-polyacrylamide gel (Novex, San Diego,
CA) and transferred to a polyvinyl difluoride membrane (Bio-Rad,
Hercules, CA). Membranes were incubated for 3 hr at room temperature
with a 1:20,000 dilution of a mouse monoclonal antibody against GFAP
(Boehringer Mannheim), followed by a 30 min incubation with a 1:3000
dilution of horseradish peroxidase-conjugated anti-mouse IgG (New
England Biolabs, Beverly, MA). The Phototope chemiluminescence system
(New England Biolabs) was used for detection of the protein recognized
by the antisera. The blots were exposed to Hyperfilm-ECL (Amersham,
Arlington Heights, IL) for varying exposure times. The protein was
detected as a band of relative molecular mass of 51 kDa. After blot
visualization, the membranes were stripped with ImmunoPure IgG Elution
Buffer (Pierce, Rockford, IL) for 3 hr at room temperature and then
blotted with a 1:20,000 dilution of the antibody to the housekeeping
gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Chemicon,
Temecula, CA) as a control. The blots were visualized as described
above. The immunoreactive band densities were quantified with a CCD
camera and densitometry using National Institutes of Health IMAGE
software. The GFAP-immunoreactive bands were standardized to the bands
immunoreactive for GAPDH (relative molecular mass of 36 kDa), and the
data are expressed as a ratio of GFAP optical density to GAPDH optical density.
Statistical analysis
Measurements for the ARC and VMN were done in the same brain and
are therefore not entirely independent of each other. However, an ANOVA
that treated each brain area as a repeated measure would fail to detect
the significant effects of hormone treatment that occur in only one of
the two areas. Thus, the comparison of the number of branches on
primary dendrites and the spine density between the four treatment
groups was analyzed by a two-way ANOVA with region and treatment
as factors. All ANOVAs were followed by a Newman-Keuls
post hoc test to determine significance between the groups.
The regional comparison of dendritic length from the ARC and VMN as
well as GFAP-IR process length in the ARC from the four treatment
groups was analyzed by a one-way ANOVA. The data on the frequency
distribution of the different classes were analyzed by chi-square
( 2). GFAP expression in the ARC and VMN assayed by
Western blotting was analyzed by Student's t test. All
statistical tests were conducted using the GB-STAT
program on a Macintosh computer.
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RESULTS |
Neuronal morphology
Incubation of PN2 brains in Golgi-Cox solution for 20 d
yielded ~15-20 well-impregnated neurons per slice in the ARC and
20-25 well-impregnated neurons per slice in the VMN. Neurons in the ARC appeared as bipolar cells with approximately two to three primary
dendrites that branched occasionally. In contrast, the neurons in the
VMN appeared much more complex with a more triangular-shaped soma and
approximately four to five primary dendrites with several degrees of
higher order branches.
The total length of dendrites in the VMN and ARC was not influenced by
the hormonal status of the animal [F(1,46) = 0.78 and F(1,46) = 0.13, respectively].
However, the mean dendritic length of neurons when pooled from the
treatment groups was 19% longer in the VMN (238.3 ± 5.9 µm)
than that in the ARC [192.5 ± 6.9 µm;
F(1,53) = 24.96; p < 0.0001].
In contrast to the lack of a hormonal treatment effect on dendritic
length, there was a significant effect on the number of branches on
primary dendrites [F(1,46) = 10.63;
p < 0.0001]. Post hoc analysis revealed
that only neurons in the VMN responded to testosterone with an
increased branching; females and castrate males had 43 and 57% fewer
branches, respectively, on their VMN dendrites than did males and
females plus TP (p < 0.01; Figs.
3,
4A,C).
There was no effect of hormonal status on dendritic branching in the
ARC. However, the main effect of treatment on spine density was in the
ARC [F(1,46) = 7.62; p < 0.001]; females and castrate males had 37% more spines on their
dendrites than did males and females plus TP (p < 0.01; Figs. 3, 4B,D). There was
no effect of treatment on the spine density in the VMN
[F(1,46) = 1.10; p > 0.05].
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Figure 3.
Camera lucida drawings of ARC and
VMN dendrites from the hormonally treated groups.
Drawings represent individual primary dendrites impregnated with
Golgi-Cox from the ARC (A) and
VMN (B) across four treatment
groups. ARC dendrites from the groups with high levels
of circulating testosterone (males and females + TP)
have significantly fewer spines on their dendrites than do groups with
low levels of testosterone (females and castrate males). Dendrites in
the VMN have significantly more branch points in groups
with high levels of circulating testosterone than do the groups with
lower levels. Scale bars, 10 µm.
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Figure 4.
Effects of neonatal hormonal manipulation on
neuronal morphology in the ARC and VMN.
Neuronal morphology was assessed using the Neurolucida system to
reconstruct Golgi-Cox-impregnated neurons in the hypothalamus of PN2
rats. Approximately 10-20 dendrites per animal in six to eight animals
per group were analyzed. Data are the mean (± SEM) of the number of
branches on primary dendrites and the spine densities from the four
treatment groups. A, C, The presence of
gonadal steroids affected the number of branches on primary dendrites
in the VMN but not in the ARC.
B, D, Conversely, the spine density of
dendrites in the ARC but not in the VMN
was affected by the gonadal steroids. Groups sharing the same
letters (a-d) in the individual graphs are
significantly different from each other (ANOVA, p < 0.01).
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Astrocytic morphology
GFAP-IR was detectable in both the ARC and VMN. However,
there were clear differences in the pattern of GFAP-IR between the two
regions in that the staining in the VMN was more diffuse and had the
appearance of radial glia. Only a few differentiated astrocytes were
observed in the VMN at PN2, and these were not readily amenable to
quantification. These observations suggested that the radial glia had
not yet differentiated into astrocytes and may express both vimentin, a
marker for immature astrocytes, and GFAP as intermediate filament
proteins (Schnitzer et al., 1981). In contrast, the GFAP-IR cells in
the ARC have a range of appearance from fully stellate cells with bushy
processes to simple bipolar cells with only a few processes. There were
no radial glia-like cells immunoreactive for the GFAP antigen in the
ARC at PN2.
In addition to the regional differences, the morphology of GFAP-IR
astrocytes in the ARC was affected by hormonal manipulations, whereas
the astrocytes in the VMN did not appear to be affected qualitatively.
This is in part because there were too few differentiated cells to
measure accurately in the VMN. In the ARC, GFAP-IR processes are 40%
longer in gonadally intact males and females injected with TP than in
females or castrate males [F(1,16) = 46.75;
p < 0.01; Figs. 5,
6A]. The GFAP-IR
astrocytes in the ARC were also categorized by class according to their
degree of differentiation. In intact males and females injected with
TP, ~50% of the total GFAP-IR astrocytes were class IV or fully
differentiated, and ~5-10% were class I or undifferentiated
astrocytes. In contrast, the ARC of castrate males and intact females
was almost exactly the opposite, being composed of 50% class I and
<15% class IV GFAP-IR astrocytes (2 = 162.4;
p < 0.01; Figs. 5, 6B).
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Figure 5.
Representative photomicrographs (20×)
of ARC GFAP-IR astrocytes across different hormonal treatments. Males
and females injected with TP have a greater frequency of
fully differentiated GFAP-IR astrocytes when compared with females and
castrate males. Scale bar, 50 µm.
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Figure 6.
Analysis of ARC
GFAP-IR astrocytes. A, Effects of
neonatal hormonal manipulation on GFAP-IR
process length. The mean distance that processes extended from the
astrocyte soma was measured in 28-40 cells per animal and four animals
per group. Increased process length is correlated with increased
differentiation. Data are the mean (± SEM) of
GFAP-IR process length in the
ARC of four treatment groups in PN2 rats. Groups sharing
common letters (a-d) are significantly
different from each other (ANOVA, p < 0.01). The
process length was significantly longer in intact males and in females
injected with TP compared with castrate males and intact
females. B, The morphological appearance of astrocytes
in the PN2 ARC. Astrocytes were categorized into four
classes (see Materials and Methods; Fig. 5), and the percentage of each
in the four treatment groups was quantified. There was a significant
difference in the distribution of cell classes with intact males and
females injected with TP having a higher frequency of class IV cells
that represent the greatest degree of differentiation. Conversely,
castrate males and intact females have more class I cells that lack a
high degree of differentiation (2, p < 0.01).
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GFAP protein levels
Using computerized densitometry, we detected a significantly lower
level of GFAP immunoreactivity in the protein from the VMN of PN2 rats
compared with that in the protein from the ARC of the same animals
(n = 7). The ratio of the optical density of the GFAP-
and GAPDH-immunoreactive bands from the ARC was ~100× more dense
than the optical density ratio of the bands from the VMN
(t = 3.05; df = 12; p < 0.02;
Fig.
7A,B.).
The immunoreactive optical density of the housekeeping protein GAPDH
was not significantly different between the ARC and VMN. GFAP and GAPDH
were represented as immunoreactive bands at 51 and 36 kDa,
respectively.
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Figure 7.
Western blot analysis. A, Four
representative electrophoretic lanes from an immunoblot
of tissue micropunches from the VMN (lanes
1, 2) and the ARC (lanes
3, 4) of two animals. Lanes
1 and 3 and lanes 2 and
4 contain tissue from the same animal. A total of 5 µg
of total protein was loaded onto each lane. The blot was
probed with a mouse monoclonal antibody to GFAP that
recognized an appropriate band at 51 kDa and with a mouse monoclonal to
GAPDH that recognized the appropriate 36 kDa band.
B, Levels of GFAP in the
VMN and ARC. Data are represented as a
ratio of the optical density of the GFAP-immunoreactive
band to that of the GAPDH-immunoreactive band. Levels
were significantly higher in the ARC compared with the
VMN of PN2 rats (p < 0.02, t test).
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DISCUSSION |
Analysis of neuronal and astrocytic morphology in the ARC and VMN
during postnatal development has revealed a complex set of interactions
that are both hormonally regulated and regionally specific. One of the
most striking observations was that in the ARC of animals with high
circulating levels of testosterone there was a coincident change in
neuronal and astrocytic morphology in response to steroids. In both
gonadally intact males and females injected with TP, there was an
increase in astrocyte process length and the frequency of fully
differentiated astrocytes. At the same time in animals with the same
hormonal profile, there was a decrease in dendritic spine density. The
reverse was true in the absence of high levels of testosterone; ARC
dendrites had an increased dendritic spine density, and the astrocytes
were less differentiated with shorter processes. In contrast, in the
VMN there was no effect of TP on dendritic spine density. However,
neurons in the VMN of males and of females injected with TP had a
higher degree of branching when compared with oil-injected females and
castrate males. Furthermore, there was no effect of TP on the
morphology of astrocytes in the VMN in these animals. In fact, there
appeared to be regional differences in astrocytic cell morphology
between the ARC and VMN in that differentiated astrocytes were
virtually nonexistent at this age in the VMN regardless of hormonal
status, a conclusion supported by the observation of extremely low
levels of GFAP in the VMN compared with the ARC. Unrelated to the
hormonal effects, developing neurons in the VMN had longer dendrites
than neurons in the ARC.
To our knowledge this is the first demonstration of coordinated
morphological plasticity in neurons and astrocytes in developing rat
brain in vivo. Several in vivo studies of adult
rat hypothalamic nuclei demonstrate neuronal plasticity in response to
alterations of the hormonal milieu in which the neural substrates in
both male and female adult rat ARC and VMN undergo dramatic structural modifications (Olmos et al., 1989; Frankfurt et al., 1990; Segarra and
McEwen, 1991; Danzer et al., 1998). A major component of neuronal plasticity appears to be changes in dendritic spine density, and estrogen has been found to influence the dendritic spine density in
adult hippocampus and VMN (Frankfurt et al., 1990; Gould et al., 1990;
Frankfurt and McEwen, 1991; Segarra and McEwen, 1991; Woolley and
McEwen, 1992). Dendritic spines are the site of excitatory synapses,
and analyses at the electron microscopy level demonstrate that
hormonally induced spines result in increased numbers of synapses
(Woolley and McEwen, 1992; Woolley et al., 1996). Thus, sexually
dimorphic dendritic spine densities in the ARC of PN2 rats may provide
the synaptic framework for sexually dimorphic neuroendocrine secretions
and behaviors exhibited by adult rats.
Neuronal-glial interactions
Neuronal-glial interactions within the CNS begin during
early stages of fetal development and extend throughout the lifespan of
adult rats, with glia playing different roles at different stages.
During neurogenesis and early development, radial glia provide a
scaffold for migrating neurons (Rakic, 1972). These radial glia serve
as precursors to the more mature astrocytes. In vitro
studies suggest that astrocytes play an active role in determining
region-specific patterns of synapse formation in developing brain, as
well as participating in synaptic plasticity in adult CNS. For example,
Le Roux and Reh (1995) have demonstrated that embryonic cortical
neurons developed three times more primary dendritic outgrowths when
cultured with astrocytes derived from the cortex, retina, and olfactory
bulb than on glia derived from the mesencephalon, striatum, or spinal
cord of postnatal rats. Moreover, they suggest that this effect is
mediated by a diffusible factor because the glia-conditioned media
elicited the same results. Alternatively, nondiffusible factors from
astrocytes may also mediate changes in neuronal morphology. In contrast
to radial glia, mature astrocytes synthesize extracellular matrix
molecules such as tenascin and chondroitin sulfate proteoglycans that
are known to be nonpermissive to neurite outgrowth (McKeon et al., 1991; Faissner and Steindler, 1995).
A hallmark of astrocyte maturation is increased synthesis of GFAP
(Eng, 1985; Condorelli et al., 1990; Eng and Ghirnikar, 1994). The
intermediate filaments of astrocytes are composed of the CNS-specific
GFAP and the less cell-specific filament protein vimentin (Bignami et
al., 1972). Vimentin is the major cytoskeletal protein expressed in
early astrocytic development (Dahl et al., 1981; Schnitzer et al.,
1981). As development progresses, radial glia are believed to be
transformed into stellate process-bearing astrocytes (Schmechel and
Rakic, 1979; Ramon y Cajal, 1995) that are marked by an accumulation of
GFAP (Culican et al., 1990), and stellation of astrocytes is generally
considered a characteristic of fully differentiated astrocytes both
in vivo and in vitro (Hatten, 1983; Grasser and
Hatten, 1990). Our analysis of regional GFAP expression by Western
blots, in combination with our visual observations, suggests that ARC
astrocytes exhibit a greater degree of maturation than do astrocytes in
the VMN, which may be the consequence of an intrinsic developmental
program. Thus, the lack of steroid-induced neuronal branching in the
ARC may be attributable to the presence of mature and/or maturing
astrocytes resulting in a nonpermissive environment for dendritic growth.
Steroid-induced neuronal-glial interactions
In the adult rat hypothalamus, astrocyte morphology changes
in response to a specific stimulus such as steroids, and the resulting morphology influences neighboring synapses (Theodosis and Poulain, 1993; Garcia-Segura et al., 1994b). Work by Garcia-Segura and colleagues demonstrates the role of neuronal-astrocytic interactions on synaptic remodeling of adult female rat ARC over the estrous cycle.
When estrogen levels are high on the day of proestrus, glia in the ARC
increase their surface area (Garcia-Segura et al., 1994a). Electron
microscopy studies suggest that extended astrocytic processes decrease
inhibitory synaptic inputs by ensheathing ARC neurons (Garcia-Segura et
al., 1994b). This "phasic synaptic remodeling" of the ARC in
response to estrogen is sexually dimorphic. Brains masculinized by
gonadal steroid exposure during the critical developmental period of
brain sex differentiation no longer exhibit this plasticity
(Garcia-Segura et al., 1994b). The current data suggest that early
differentiation of ARC astrocytes after exposure to testosterone and
its metabolite estrogen may be causally related to the loss of
plasticity in the adult male.
The majority of sex differences in the rodent brain are caused by
local conversion of testosterone to estrogen by the p450 aromatase
enzyme (Naftolin et al., 1975; McEwen et al., 1977; Lephart, 1996) that
is localized in neurons and does not appear to be present in astrocytes
(Martini and Melcangi, 1991). We have confirmed that neonatal ARC glia
are also responding to locally synthesized estrogen, rather than to the
testosterone itself (Mong and McCarthy, 1998). However, whether
estrogen is acting directly on neurons, astrocytes, or both is unknown.
Steroids, such as estrogen, principally act via intracellular receptors
that translocate to the nucleus after binding and alter gene
transcription. Estrogen receptor protein and mRNA have been reported in
cultured astrocytes from several brain regions (Jung-Testas et al.,
1992; Santagati et al., 1994; Murphy et al., 1998) including the
hypothalamus (Ma et al., 1994; Ma and Ojeda, 1997), and estrogen
receptors have been immunocytochemically detected in guinea pig glia in the preoptic area and the median eminence (Langub and Watson, 1992).
However, there has been no clear in vivo demonstration of
any gonadal steroid receptors in hypothalamic astrocytes of neonatal
rats (Garcia-Segura et al., 1996). Moreover, neonatal rat hypothalamic
astrocytes in vitro differentiate in the presence of
estrogen only when cocultured with hypothalamic neurons (Torres-Aleman et al., 1992), and the presence of neurons in astrocytic cultures has
been shown to reverse the effect of estrogen on GFAP mRNA synthesis
(Stone et al., 1998). Therefore, the cellular site of estrogen action
remains unclear. In view of this, we envision three possible scenarios
for estrogen-induced changes in neuronal-astrocytic morphology in the
neonate. First, in the ARC, neurons convert testosterone into estrogen
where it initiates a signaling cascade that transduces this signal to
the surrounding astrocytes to increase differentiation. A signal
from the differentiated astrocytes in turn attenuates neuronal
formation of dendritic spines. The decrease in spine formation in the
ARC may be the result of a physical blockade by astrocytic processes or
by some diffusible factor that is released from glia and signals back
to the neuron. In contrast, either astrocytes in the VMN are not
sufficiently mature to respond to a differentiating signal from the
neurons, or neurons of the VMN do not respond to estrogen with the
production of such a signal. Regardless, the lack of astrocyte
differentiation in the VMN may create a permissive environment for
estrogen-induced dendritic growth and branching. The second possibility
is that estrogen acts directly on neurons in both the ARC and VMN to
influence changes in neuronal morphology in a regionally specific
manner and mediates the release of an astrocytic differentiating factor in the ARC. Third, it is possible that estrogen is acting directly on
ARC astrocytes to induce their differentiation either via steroid receptors or via other nontraditional pathways and that changes in
neuronal and astrocytic morphology are independent of each other. Our
future experiments will distinguish between these scenarios.
| |
FOOTNOTES |
Received Aug. 6, 1998; revised Oct. 29, 1998; accepted Dec. 2, 1998.
This work was supported by National Science Foundation Grant
IBN-9511328 to M.M.M. We would like to thank Drs. Greg Ball, Anne
Murphy, and Scott Thompson for helpful comments on this manuscript.
Correspondence should be addressed to Jessica A. Mong, Department of
Pharmacology, University of Maryland, 655 West Baltimore Street,
Baltimore, MD 21201.
| |
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Top
Abstract
Introduction
