Susana Isabel Ferreira da Silva de Sá ESTROGÉNIOS E ...

SYNAPTIC PLASTICITY IN THE VM NUCLEUS
number (Frankfurt et al., 1990; Madeira et al., 2001) in
response to high levels of estrogen, and these changes
occur reversibly every 4–5 days. To modulate neuronal
plasticity in the VMNvl of the female rat, estrogen might
act through classic nuclear receptors and/or via nongenomic
mechanisms. Actually, it is known that 30% of
the neurons in the VMNvl concentrate estrogen (Morrell
and Pfaff, 1983) and that some of the effects of this hormone,
namely, those noticed in dendritic spine density,
are not confined to neurons that express the estrogen
receptor (Calizo and Flanagan-Cato, 2000). The finding
that some of the effects of estrogen are blocked by surgical
deafferentation of the VMN (Nishizuka and Pfaff, 1989)
suggests that they are mediated by VMN afferents, inasmuch
as most of them express estrogen receptors (Heimer
and Nauta, 1969; Saper et al., 1978; Simerly and Swanson,
1988; Canteras and Swanson, 1992; Canteras et al.,
1992, 1995). This possibility led us to examine whether
the pattern of connectivity of VMNvl neurons changes
over the estrus cycle. Thus, we estimated the number and
the size of the synaptic contacts established upon the
perikarya and the dendritic trees, including their spines,
of VMNvl neurons in proestrus and in diestrus rats, i.e., at
phases of the estrus cycle that typically show opposite
hormonal profiles.
The VMN has long been thought of as a sexually dimorphic
nucleus, and there are descriptions of male–female
differences in its volume (Matsumoto and Arai, 1983; Madeira
et al., 2001), neurochemistry (reviewed in Madeira
and Lieberman, 1995), spine density (Madeira et al.,
2001), and density of axodendritic spine and shaft synapses
(Matsumoto and Arai, 1986b; Miller and Aoki,
1991). There is evidence that the sex-related differences in
the density of axodendritic synapses result from the organizational
effects of sex steroid hormones during development
(Matsumoto and Arai, 1986a; Miller and Aoki, 1991),
and that the adult steroid environment in females is a
determining factor of the magnitude of the morphological
sex differences in the VMN (Madeira et al., 2001). In fact,
the sex differences in the volume of the VMN and in
dendritic spine density are maximal when females are in
diestrus and in proestrus, respectively, whereas the sexrelated
differences in neuronal size, namely, in the length
of the terminal dendritic branches, become apparent only
at certain phases of the estrus cycle. Thus, even though
male–female differences have been noticed in the numerical
and areal density of synapses, parameters which are
sensitive to variations in the volume of the reference
space, it still remains unknown whether or not the absolute
number of synapses in the VMNvl is sexually dimorphic.
The present work was also designed to evaluate this
issue by estimating in parallel the number and the size of
synaptic contacts in the VMNvl of male and female rats.
MATERIALS AND METHODS
Animals
Young-adult male and female Wistar rats were maintained
on a 12-hour light/dark cycle (lights on at 0700) and
ambient temperature of 22°C, with food and water available
ad libitum. The estrus cycle was monitored by daily
inspection of vaginal cytology for at least 3 weeks before
killing. Only regularly cycling rats were used in the
present study. Animals were killed at 3 months of age
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between 1400 and 1600 hours. The group of males consisted
of six animals, and the group of females of six rats
in proestrus and six rats in diestrus day 1 (diestrus). All
studies were performed in accordance with the European
Communities Council Directives of 24 November 1986
(86/609/EEC) and Portuguese Act 129/92.
Hormonal determinations
Prior to perfusion, blood samples (500 l) were taken
directly from the heart into Eppendorf tubes. The serum
was separated by centrifugation and stored at –20°C until
the time of assay. The concentrations of 17 -estradiol
were determined by radioimmunoassay techniques using
kits that were purchased from MP Biomedicals (Irvine,
CA).
Tissue preparation
Animals were anesthetized with 3 ml/kg of a solution
containing sodium pentobarbital (10 mg/ml) and chloral
hydrate (40 mg/ml) given i.p. and killed by transcardiac
perfusion of a fixative solution containing 1% paraformaldehyde
and 1% glutaraldehyde in 0.12 M phosphate
buffer, pH 7.2. The brains were removed from the skulls,
weighed, and immersed in the same fixative solution for 1
hour. After postfixation they were bisected sagittally
through the midline.
The right and left hemispheres were transected in the
coronal plane through the posterior border of the optic
chiasm. After removal of the occipital poles, they were
mounted on a Vibratome with the rostral surface up.
Forty-m-thick coronal sections were cut through the tuberal
region of the hypothalamus, mounted on slides,
stained with thionin, and observed under the optic microscope.
When the VMN was first identified as being formed
by two well-defined clusters of cells, the dorsomedial and
the ventrolateral (VMNvl) divisions, separated by a cellpoor
central zone (Bleier et al., 1979; Simerly, 1995; Madeira
et al., 2001), Vibratome sections of alternate thickness
(40 m and 500 m) were obtained. The 40-m-thick
sections were mounted on slides and stained with thionin
for precise identification of the boundaries of the VMN and
of its dorsomedial and ventrolateral divisions (Fig. 1).
Based on tissue landmarks visible in these sections, the
adjacent 500-m-thick sections were dissected under microscope
observation to isolate the VMNvl (Fig. 1C,D). The
blocks of tissue containing the VMNvl were then processed
for electron microscopy. For this purpose, they were
postfixed for 2 hours in a 2% solution of osmium tetroxide
in 0.12 M phosphate buffer, rinsed in 25% ethanol, and
dehydrated through graded series of ethanol solutions
(90%, 95%, and 100%). The blocks were then stained in 1%
uranyl acetate for 1 hour at room temperature at the 70%
alcohol stage. After passage through propylene oxide, the
blocks were embedded in Epon according to the isector
method (Nyengaard and Gundersen, 1992). Accordingly,
embedding took place in molds containing spherical cavities;
the resulting spherical embedded blocks were rolled
and thereafter reembedded in Epon.
From each of the four blocks obtained per animal, eight
serial 2-m-thick sections of the VMNvl were cut, which
provided a total of 32 serial semithin sections per animal.
Each semithin section was placed on a drop of distilled
water on a gelatin-coated microscope slide, dried on a
slide-warming plate at 60°C, stained with Toluidine blue,
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