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Localization of cells retaining 3H-estradiol in the forebrain of rabbits.

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Localization of Cells Retaining 3H-estradiol in the
Forebrain of Rabbits '
Department of Anatomy, University of Illinois at the Medical Center,
1853 W. Polk Street, Chicago, Illinois 60680
Ovariectomized rabbits received "-estradiol via an ear vein and
were killed one hour later. Autoradiograms were prepared and exposed up to six
months. Labeled cells, as indicated by many silver grains over the nucleus of a
neuron, were found in many nucIei of the brain. Thus, the bed nucleus of the
stria terminalis had labeled cell bodies. The stria terminalis leads into the medial
preoptic area where great numbers of cells concentrated the estrogen. Farther
into the hypothalamus the labeled cells were numerous in the ventromedial and
arcuate nuclei. Other locations with labeled cells were the lateral septa1 nucleus
and nucleus accumbens septi, the periventricular preoptic nucleus, anterior hypothalamic nucleus, nucleus supraopticus diffusus, posterior hypothalamic nucleus,
and premammillary nucleus. The labeled cells could be followed into the central
gray surrounding the aqueduct of Sylvius. The amygdaloid nuclei, and in particular the medial amygdaloid nucleus, had labeled cells as did the most ventral
posterior part of the hippocampal cortex. The results are discussed in comparison
with those in the rat. and with reference to physiologic data.
Several studies have confirmed the autoradiographic localization of tritiated estradiol in cells in the brain of the rat (Stumpf,
'68; Pfaff, '68; Anderson and Greenwald,
'69; Attramadal, '70). These studies can
be correlated in part with reports on hormone secretions and behavior after lesions
or steroid implants. The implantation studies provide localized concentrations of hormone in the brain so that physiological end
points can be observed and related to a
hormone-brain interaction. These studies
have demonstrated the need to consider a
series of interconnected structures in the
brain that are related to complex behavioral and physiologic functions (Rodgers
and Law, '68).
In this study, the rabbit was investigated
because, in some respects, it has served as
a contrast to the rat in the control of reproduction. Thus, the rabbit is a reflex ovulator, and the area for control of reproductive behavior appears to be in the posterior
hypothalamus (Palka and Sawyer, '66).
Because of the species differences, it was
felt worthwhile to study the location of
neurons which concentrate estradiol in the
ANAT.REC.,181: 287-292.
New Zealand white rabbits (from Scientific Small Animals) weighing 4-5 pounds
and approximately two months of age were
housed under 14 :10 light: dark, with constant temperature. A sample of estradiol,
2,4,6,7-tritiated (New England Nuclear,
106 C/mM) was chromatographed on
ITLC (Hexane:Ethyl acetate, 10:2) to assure its purity. One week after ovariectomy, the radioactive estrogen, 100 pc, was
injected via an ear vein in a 20% EtOH
in normal saline solution. One hour after
the injection, the rabbit was killed with an
overdose of Nembutal (1.V.) and quickly
decapitated. The brain was removed from
the skull, frozen over liquid nitrogen and
stored in liquid nitrogen until sectioning.
For the autoradiography, acid cleaned
slides were coated with Kodak NTB-3 emulsion. Sections cut at 4-6
at - 18" to
- 20°C, were then picked up on a precoated slide. The sections melted onto the
slide and dried quickly. The slides were
stored with drierite at 4°C for six months.
Received March 8, "74. Accepted
. . 15,. '74.
1 This work has been supported by a grant from the
NationaI Institute of Child Health and Human Development, NIH, HD-5865.
Dektol developer ( 2 min. ) and Kodak acid
fixer (5 min.) were used to process the
film. The sections were stained with hematoxylin. Positive and negative chemography
in the autoradiograms were checked by
appropriate exposure of film to non-radioactive tissues and are not a problem.
Similar experiments were performed with
2,4,6,7-3H estrone in ovariectomized rabbits (New England Nuclear, 106 C/mM).
The sections were systematically scanned
at 400X to identify and locate labeled cells.
A cell was considered labeled if at least
4-5 silver grains were over the nucleus.
The atlases of Sawyer et al. ('54), Urban
and Richard ('72) and Fifkovh and MarGala ('67) were used to identify nuclear
groups. Numbers of labeled cells in autoradiograms of hypothalamic and preoptic
areas were determined in one rabbit to
gain an impression of the distribution of
labeled cells.
Cells concentrating radioactivity were
found in all amygdaloid nuclei, but subjectively, the medial amygdaloid nucleus
had the greatest number. Labeled cells
could be seen in the bed nucleus of the
stria terminalis. This tract projects into
the medial preoptic area where the number
of labeled cells per unit area was quite
large (figs. 2, 4 ) . The ventrally placed
periventricular nucleus of the preoptic
area, beside the supraoptic recess of the
third ventricle, also had many labeled cells
(fig. 2 ) .
In the hypothalamus, many labeled cells
were found in the arcuate nucleus imme-
Fig. 1 Representative autoradiogram showing
cells labeled with tritiated-estradiol. Arcuate nucleus. x 635.
diately above the median eminence (fig. 1).
A few labeled cells at the ventral lateral
border of the arcuate nucleus were in the
median eminence. Immediately behind the
optic chiasma the nucleus supraopticus
diffusus had numerous labeled cells. This
area was just above the median eminence
(fig. 2). Scattered labeled cells were also
found in the anterior hypothalamic nucleus and adjacent periventricular nucleus
as well as a few throughout the entire ventromedial nucleus. Between the arcuate
nucleus and the ventral border of the ventromedial nucleus many labeled cells were
found. The major concentration of cells in
the ventromedial nucleus was on the ventral surface and continued up the lateral
border to the area immediately below and
medial to the fornix, thus forming an arc
of labeled cells (fig. 2). Between ventromedial and mammillary nuclei, cells of the
premammillary nucleus also accumulated
A few labeled cells were found in the
posterior hypothalamic nucleus and these
sparsely distributed cells could be followed
into the central gray of the midbrain
(which was as far caudally as sections
were cut). There were also labeled cells in
the lateral septa1 nucleus and the nucleus
accumbens septi. In the most ventral extent of the hippocampal formation (posterior hippocampus), and probably restricted
to the CA1 area of Lorente de N6 ('34),
there were many labeled cells in a row of
large neurons (fig. 3). Furthermore, the
cells retaining estradiol tended to be in the
pyramidal layer and placed near the stratum oriens.
Figure 4 shows the numbers of labeled
cells in several sections throughout the
preoptic area and hypothalamus of one
brain. The distribution of the labeled cells
was quite similar to what has been reported
for rats (Anderson and Greenwald, '69),
except the numbers of cells immediately
behind the optic chiasma does not drop
near zero due to the numerous cells in the
nucleus supraopticus diffusus. Posterior to
the pituitary stalk the majority of labeled
cells were those in the arcuate nucleus.
The injection of tritiated estrone did not
result in heavily labeled cells in the amygdala or hypothalamus. However, a very few
labeled cells were found in the posterior
Fig. 2 Coronal secCions of rabbit hypothalamus. Dots indicate locations of labeled cells,
The lateral ventricles and third ventricle are shown in cross hatching.
Abbreviations: A, arcuate nucleus; AC, anterior ccmmissure; CC, corpus callosum; DHA,
dorsal hypothalamic area; DMH, dorsomedial hypothalamic nucleus; F, fornix; FF, fimbria
of fornix; HIP, hippocampus; LHA, lateral hypothalamic area; LPO, lateral preoptic area;
MPO, medial preoptic area; OC, optic chiasm; OT, optic tract; PHA, posterior hypothalamic
area; PMA, premammillary area; PPO, preoptic periventricular nucleus; PV, paraventricular
nucleus; SCH,suprachiasmatic nucleus; SO, supraoptic nucleus; SOD, supraopticus diffusus
nucleus; SP, septum; VMH, ventromedial hypothalamic nucleus.
Fig. 3 Ventral hippocampus. Inked black dots
show the location of a row of labeled cells on the
deep side of the pyramidal layer. x 13.
hypothalamic area. These cells were not
numerous and the assurance of the identification deserves further support.
In in vitro studies, hypothalamic tissue
Fig. 4 Number of labeled cells in cross sections of rabbit hypothalamus, closed circles. Number of cells in arcuate nucleus only, shown with
Xs. Animal killed 1 hour after IV injection of
Abbreviations: AC, anterior commissure; OC,
optic chiasm.
from rabbits did not metabolize 'H-estradiol
to estrone or estriol (Chader and Villee,
'70). This corresponds with reports in other
species in which in vivo experiments
found the majority of the radioactivity detected was 'H-estradiol (Wade and Feder,
'72). Thus, the author assumes that at
least the majority of radioactivity detected
in this experiment was associated with
estradiol. In a series of papers on the rabbit, the area posterior to the hypophyseal
stalk was found important for the control
of estrous behavior, for the reflex secretion
of ovulating hormone( s ) and for feedback
inhibition of LH by estrogen (Sawyer, ’59;
Davidson and Sawyer, ’61; Kanematsu and
Sawyer, ’63; Palka and Sawyer, ’66). It was
anticipated from the contrast of these findings with those in rats, where these “centers” are more anteriorly localized, that
there might also be some difference in the
pattern of cells which accumulate estradiol, However, with the exception of species differences in shapes and dimensions
of nuclei, the cells that retain estrogen
were in virtually the same locations as in
the rat. That is, a “system” of neurons that
bind estrogen forms a continuum from
amygdaloid nuclei through cells in the bed
nucleus of the stria terminalis to medial
preoptic and hypothalamic nuclei.
Palka and Sawyer (’66), using implants,
localized the most estrogen sensitive area
for induction of sexual behavior in the posterior ventromedial nucleus - premammillary area, up to 1 mm from midline but
not in midline or third ventricle. This same
area had numerous, heavily labeled cells
which may be important in the neuronal
circuits eliciting the estrous response. Dorsal to this area there were a few labeled
cells in the hypothalamic posterior and
dorsomedial nuclei, but the number of cells
was not great and they were sparsely distributed.
As was previously reported for the rat,
there were cells in the hippocampus which
accumulate estradiol (Stumpf and Sar,
’69). The cells show a very restricted distribution which appears to correlate well
with the CA1 area of the ventral (posterior) hippocampus (Lorente de N6, ’34).
The posterior part of field CA1 projects to
sites different from those to which the anterior part projects. The posterior CA1 field
has connections to septal nuclei and diagonal band nuclei, while the anterior CA1
field does not project to these structures
(Raisman et al., ’66). The labeled hippocampal cells were large and appear to correspond to the deep pyramidal cells of
Lorente de N6 (’34), but may possibly be
basket cells as described by Raisman et al.
(’65, p. 967). Basket cells form inhibitory
synapses with pyramidal cells (Anderson
et al., ’63), particularly in the superficial
layer of pyramids and seem to have less
input to the deep pyramids (Lorente de N6,
’34). Labeled cells of the hippocampus and
septal nuclei form a second “system” of
labeled neurons.
Gallo et al. (’71), in the rat, have demonstrated effects of ventral hippocampal
and subiculum stimulation on gonadotropin secretion and multiple unit activity in
the arcuate nucleus. They also found, in
multiple unit recording experiments in the
ventral hippocampus-subiculum, that injected LH would reduce recorded activity
in one-half of the sites explored (Gallo et
al., ’72). The labeled cells in the present
experiment may be slightly displaced laterally from the subiculum-LH responsive
area identified by these authors. According
to Nauta (’56) and Raisman et al. (’66),
the medial corticohypothalamic tract originates exclusively from the caudal onethird of the rat subiculum. This tract
projects to the arcuate nucleus of the
hypothalamus .
The biochemistry of binding and specific
cellular effects of estrogen have been
most clarified in the uterus (Jensen and
DeSombre, ’73). In brain, the localization
by a cell may result in an excitatory or
inhibitory influence on that cell, and that
cell, via a short axon may have local actions or, via long axons, may act upon distant neural structures. Thus, correlating
specific cells which accumulate estradiol
with specific functions, behavioral or hormonal, is by suggestion only. The labeled
cells shown for the rabbit have a wide distribution through the preoptic area and
hypothalamus. Physiological studies, on
the other hand, indicate fairly restricted
localization to the posterior baso-medial
hypothalamus for reproductive function.
Clearly, further investigation is needed on
the role of the estrogen binding neurons in
the preoptic area as well as the labeled
cells of the amygdala and hippocampus.
The author thanks Mrs. Zanet F. Gill for
excellent technical assistance.
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estradiol, retaining, rabbits, localization, forebrain, cells
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