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The Journal of Neuroscience, April 15, 1998, 18(8):3003-3013
Development of a Sexually Dimorphic Projection from the Bed
Nuclei of the Stria Terminalis to the Anteroventral Periventricular
Nucleus in the Rat
Leslie A.
Hutton1,
Guibao
Gu1, and
Richard B.
Simerly1, 2
1 Division of Neuroscience, Oregon Regional Primate
Research Center, Beaverton, Oregon 97006, and 2 Program in
Neuroscience, Oregon Health Sciences University, Portland, Oregon 97201
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ABSTRACT |
The principal nucleus of the bed nuclei of the stria terminalis
(BSTp) is larger in male rats and conveys olfactory information relevant for reproduction to the hypothalamus. In males, the BSTp provides a massive projection to the anteroventral periventricular nucleus of the preoptic region (AVPV), which in contrast to most sexually dimorphic nuclei contains more neurons in female rats. Injections of the anterograde tracer Phaseolus vulgaris
leucoagglutinin into the BSTp of adult female rats failed to
demonstrate the strong projection to the AVPV observed previously in
males. The ontogeny of this robust sex difference was examined by using
the axonal marker DiI. The projection from the BSTp to the AVPV is
established between postnatal day 9 (P9) and P10 in male rats and seems
to be maintained during the juvenile period. Although labeled fibers extended from the BSTp toward the preoptic region in both male and
female neonates, a similar connection with the AVPV was not apparent in
female rats at any of the ages studied, and the density of labeled
axons in the AVPV of P10 males was 20-fold greater than that of P10
females. A projection from the BSTp to the medial preoptic nucleus was
also weaker in females but was much more substantial than that to the
AVPV. These findings suggest that a sex- and region-specific activity
influences the development of the projection from the BSTp to the AVPV,
producing a sexually dimorphic architecture in pathways that convey
olfactory information to the hypothalamus.
Key words:
sexual differentiation; preoptic region; DiI; connections; dimorphic; development; bed nucleus of the stria
terminalis
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INTRODUCTION |
Sex steroid hormones exert profound
effects on mammalian forebrain development, including regulation of the
volume of sexually dimorphic nuclei, the density of certain classes of
synapses, and the number of neurotransmitter-specific neurons (for
review, see Simerly, 1990 ; Matsumoto, 1991; Segovia and
Guillamón, 1993). In addition to regulating the number of neurons
in sexually dimorphic nuclei, sex steroids seem to regulate the
development of sexually dimorphic patterns of connectivity in forebrain
regions thought to play important roles in mediating reproductive
function. Raisman and Field (1971) were the first to demonstrate a sex
difference in the density and organization of synapses in the preoptic
region of the rat; sexually dimorphic patterns of connectivity in the medial nucleus of the amygdala and in the ventrolateral part of the
ventromedial hypothalamic nucleus have also been identified (Matsumoto,
1991). However, a similar sex difference in synaptic connections was
not found in the dorsomedial part of the ventromedial nucleus
(Matsumoto and Arai, 1986), indicating that the development of sexually
dimorphic connections is highly localized and region specific.
In rodents, pheromonal olfactory information is conveyed from the
vomeronasal organ to the accessory olfactory bulb and relayed then to
the hypothalamus along a sexually dimorphic pathway comprised of
neuronal cell groups that are larger in male rats. Each of these
regions seems to influence the expression of reproductive behavior and
participate in the neural control of gonadotropin secretion (for
review, see Simerly, 1990; Segovia and Guillamón, 1993). The
principal nucleus of the bed nuclei of the stria terminalis (BSTp) (for
parcellation and nomenclature, see Ju and Swanson, 1989) is a key
component of this olfactory pathway and seems to develop under the
influence of perinatal sex steroid hormones (del Abril et al., 1987).
In adult animals, the BSTp contains high densities of neurons that
express high levels of receptors for estrogen (ER) and androgen (AR)
(Simerly et al., 1990). Moreover, the BSTp displays high levels of
steroid binding, and the majority of its neurons express ER and AR mRNA
during the postnatal period (Don Carlos and Handa, 1994;
McAbee and Don Carlos, 1996) (R. B. Simerly, unpublished
observations). Expression of androgen and estrogen receptors in the
BSTp and the AVPV seems to be sexually dimorphic in rats. AR expression
in the BSTp tends to be higher in males, whereas levels of ER
expression are generally higher in females (Simerly et al., 1990).
Because the BSTp contains high levels of the enzyme aromatase (Shinoda
et al., 1994), which converts testosterone to estradiol, either
receptor may mediate the effects of elevated levels of circulating
testosterone on the development of BSTp neurons.
The BSTp sends strong projections to hypothalamic nuclei, such as the
medial preoptic nucleus and the ventromedial hypothalamic nucleus,
known to play important roles in the regulation of reproductive behavior (Simerly, 1990). In addition, the BSTp sends strong inputs to
cell groups in the periventricular zone of the hypothalamus that are
thought to regulate hormone secretion from the anterior pituitary (Gu
and Simerly, 1997a). The anteroventral periventricular nucleus of
the preoptic region (AVPV) is of particular interest because it seems
to represent a nodal point in the neural circuits that regulate
gonadotropin secretion (Simerly, 1998). Lesions of the AVPV block
spontaneous ovulation (Wiegand and Terasawa, 1982), and neurons in the
AVPV seem to provide direct inputs to gonadotropin-releasing hormone
(GnRH)-containing neurons in the preoptic region and to
tuberoinfundibular dopaminergic neurons in the arcuate nucleus (Gu and
Simerly, 1997b) that regulate the secretion of LH and prolactin,
respectively (Fink, 1988; Neill and Nagy, 1994). Like the BSTp, the
AVPV is sexually dimorphic and contains high densities of neurons that
express estrogen receptors during the perinatal period (Don Carlos and
Handa, 1994) (Simerly, unpublished observations). In contrast to the
BSTp, which is larger in males, the AVPV appears to contain more
neurons in female rats (Sumida et al., 1993). Thus, the connection
between the BSTp and the AVPV represents a hormone-sensitive interface
between the telencephalon and diencephalon. Moreover, this connection
links two sexually dimorphic nuclei that have divergent developmental histories, a hormonally induced reduction of neurons in the AVPV of
males concomitant with an increase in the number of neurons in the BSTp
(del Abril et al., 1987; Sumida et al., 1993).
The purpose of the present study was to compare the strength of the
projection from the BSTp with the AVPV in male and female rats and to
study the development of this pathway. Anterograde transport studies
identified a robust sexually dimorphic projection in adult animals. In
male rats, the BSTp provides a massive projection to the AVPV, but in
females, the AVPV seems to receive only a weak input from the BSTp. DiI
axonal transport was used to define the temporal organization of the
development of this pathway and to determine whether this connection is
established early in both sexes and retracts in females or never
achieves in females the density of innervation that is characteristic
of male rats. Our results indicate that, in males, axons from the BSTp
reach the AVPV on postnatal day 9 (P9) and form a dense plexus of
fibers in the AVPV by P10. Although a few projection fibers from the BSTp reach the AVPV by P10 in female rats, the dense innervation pattern seen in males was not observed at any age.
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MATERIALS AND METHODS |
Phaseolus vulgaris leucoagglutinin transport
studies in adult animals. The protocols used for the adult and
neonatal studies were approved by our Institutional Committee for the
Care and Use of Animals in Research and Education, in accordance with
the guidelines of the National Institutes of Health and United States Department of Agriculture. The projection from the BSTp to the AVPV was
traced in adult rats by placing iontophoretic injections of P. vulgaris leucoagglutinin (PHAL) into the BSTp and by
immunohistochemically labeling axons containing the anterogradely
transported lectin according to methods described elsewhere (Gu and
Simerly, 1997b). Ten adult male and 10 adult female Sprague Dawley rats
(~260-310 gm) received injections of PHAL in the BSTp. Briefly, a
single iontophoretic injection of a 2.5% solution of PHAL in 0.1 M sodium phosphate buffer, pH 7.4, was made through a
stereotaxically positioned glass micropipette by applying a +4.5 µA
current, pulsed at 7 sec intervals, provided by a constant current
source (model CS4; Transkinetics, Canton, MA) for a period of 15-20
min. After a survival time of 14 d after the injection, each
animal was deeply anesthetized with tribromoethanol and perfused
transcardially with a solution of 4% paraformaldehyde in 0.1 M sodium borate buffer, pH 9.5. The brains were immediately
removed and post-fixed in the same fixative containing 10% sucrose.
Frozen sections (30 µm thick) were cut on a sliding microtome in the
frontal plane and collected through the preoptic region (one section
from every 120 µm). Additional sections were collected through the
rostral forebrain containing the BSTp and were "quick-stained" for
confirmation of the injection site location (Simerly and Swanson,
1988). Sections were incubated for 1 hr in a 1:500 dilution of rabbit
anti-PHAL that was localized with an affinity-purified goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITC) (Tago,
Burlingame, CA). One complete series of sections was processed for
immunohistochemistry with a rabbit antiserum directed against PHAL
(Dako, Carpenteria, CA) at a dilution of 1:2000. The primary antiserum
was localized by using a variation of the avidin-biotin complex system
(ABC) (Hsu and Raine, 1981; Hsu et al., 1981) with a commercially
available kit (ABC Elite Kit; Vector Laboratories, Burlingame, CA). The sections were mounted onto gelatin-coated slides and treated with osmium tetroxide to enhance visibility of the reaction product according to methods described in detail elsewhere (Simerly and Swanson, 1988). Slides were then dehydrated, coverslipped with DPX
mountant (Electron Microscopy Sciences, Fort Washington, PA), and
examined with both conventional and dark-field-transmitted light
microscopy. An adjacent series of sections was stained with thionin to
serve as a reference series for cytoarchitectonic purposes. Only cases
with injections centered within the morphological borders of the BSTp
were included in the analysis. In such cases, nearly all of the
PHAL-labeled neurons were contained in the BSTp, and the injections
were of comparable size in the male and female animals.
DiI implants in postnatal rats. Neonatal male and female
Sprague Dawley rats were anesthetized with tribromoethanol and perfused on P0, P2, P4, P6, P7, P9, P10, and P22 with a 4% paraformaldehyde solution, pH 9.5. The brains were immediately removed and stored in
fixative at 4°C until further processing. Each brain was blocked and
embedded in 1.5% agarose and sectioned from caudal to
rostral to expose the BSTp without disturbing the preoptic
region. Each brain block was stained with methylene blue to
visualize morphological features of the exposed surface of
the block, which allowed unambiguous identification of the
morphological borders of the BSTp. An insect pin was used to
place a small crystal of DiI
(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate;
Molecular Probes, Eugene, OR) into the BSTp of each brain under visual
guidance. For all implants, we selected DiI crystals that were similar
in size as judged by eye when viewed under a dissecting microscope. The
brain blocks were stored in 0.4% formaldehyde, and the DiI was allowed
to diffuse for 1 month in the dark at 37°C. The brains were then
re-embedded in 1.5% agarose, and 100-µm-thick sections through the
preoptic area were cut on a vibratome, mounted onto
poly-L-lysine-coated glass slides, and coverslipped with
10% buffered glycerol. Sections through the preoptic region were
evaluated with both conventional fluorescence and confocal
microscopy.
Evaluation. DiI-labeled material was viewed with a Leitz
diaplan epifluorescence microscope equipped with a rhodamine filter set
for viewing the orange-red DiI fluorescence. A series of 20-µm-thick sections through the preoptic area of a P10 male rat was used as a
standard reference Nissl series. Sections of this Nissl series were
used to prepare projection drawings through representative levels of
the preoptic area, and the distribution of labeled fibers in
experimental cases was charted onto this series of drawings for
illustrative purposes. The nomenclature and subdivisions of the BST are
based on that of Ju and Swanson (1989) and of Swanson (1992). In
transposing our results to the standard series, we used constant
observation of the experimental material to plot the distribution of
fibers onto the standardized drawings to achieve the most accurate
representation of the fiber distribution. Because the tissue was not
Nissl stained, we used our best estimation of the location of fibers in
the sections, and we must stress that the maps represent the fiber
distribution derived from one to two sections projected onto a
standardized drawing for each level. The drawings are not meant to
represent a quantitative evaluation of the development of the
projection but rather serve to illustrate the overall distribution of
fibers during development.
Quantitative analysis. Confocal images of DiI-labeled fibers
were collected through the AVPV and the medial part of the medial preoptic nucleus (MPNm) of P10 male and female rats by using a Bio-Rad
MRC 1000 confocal microscope. For quantification, a series of 10 adjacent optical sections were collected through the AVPV of male and
female rats along the rostrocaudle axis by using a 40× objective
[numerical aperture (NA), 1.3; working distance, 0.8-1.3 mm] and a 1 µm Z interval. Image analysis was performed by using Metamorph image
analysis software (Universal Imaging Corporation, West Chester, PA).
Maximum intensity projections of each series of confocal images were
prepared and binarized according to user-defined threshold parameters
that optimized detection of labeled fibers. The total number of pixels
in each binarized image was counted and used as an index of the overall density of labeled fibers in the AVPV.
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RESULTS |
The projection from the BSTp to the AVPV in adult male and female
rats was examined using PHAL anterograde tracing. In adult male rats,
injections of PHAL that were centered in the BSTp labeled fibers that
either pass ventrolaterally toward the medial forebrain bundle and
through the substantia innominata or pass ventromedially to innervate
structures in the preoptic region. The latter fibers form a
particularly dense plexus of labeled axons and fiber terminals in the
MPNm and AVPV (Fig.
1A,C,
respectively). Similar injections of PHAL into the BSTp of adult female
rats also labeled a plexus of fibers in the MPNm that appeared to be
somewhat less dense than was that of males (Fig.
1A,B). However, the AVPV of females contained only a few labeled fibers in marked contrast to the high
density of labeled fibers and terminals found in the AVPV of males
(Fig. 1C,D). Evaluation of the PHAL injection
sites revealed that the placement was similar when male and female
cases were compared (Fig. 2).
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Figure 1.
Dark-field photomicrographs of frontal sections
through the preoptic region that compare the distribution and density
of PHAL-labeled fibers in the AVPV (C, D)
and MPNm (A, B) of adult male
(A, C) and female
(B, D) rats. Scale bars:
A, B, 20 µm; C,
D, 50 µm.
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Figure 2.
Camera lucida drawings that show the distribution
of labeled neurons (filled circles) after
injection of P. vulgaris leucoagglutinin in the
BSTp of an adult male (top) and female
(bottom) rat. AHNa, Anterior hypothalamic
nucleus, anterior part; BST, bed nuclei of the stria
terminalis; BSTp, principal nucleus of the BST;
fx, fornix; LHA, lateral hypothalamic
area; MPN, medial preoptic nucleus;
PVHap, paraventricular nucleus of the hypothalamus,
anterior parvicellular part; SI, substantia innominata;
sm, stria medullaris; st, stria
terminalis; V3, third ventricle; SCH, superchiasmatic
nucleus; shaded grey areas, fibertracts; dashed
lines, borders of nuclei.
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To examine the development of the sexually dimorphic projection from
the BSTp to the AVPV, we used DiI axonal labeling. Brains with DiI
crystals centered in the BSTp were obtained from P2 (n = 3), P4 (n = 3), P7 (n = 6), P9
(n = 3), and P10 (n = 4) male rats. In
these cases, the crystalline deposits of DiI were confined to the BSTp
and were ~25-35 µm in diameter. Slight differences in crystal size
probably do not account for the differences we observed between
postnatal ages and sexes because even a small DiI crystal placed in the
tissue probably saturates the area adjacent to the implant with dye.
When the DiI crystal was centered outside the morphological borders of
the BSTp, a distinctly different pattern of labeling was observed with
very few fibers entering the medial preoptic area, suggesting that
diffusion of DiI beyond the morphological borders of the BSTp did not
contribute to the labeling observed in the AVPV or MPNm. When the DiI
crystal was centered in the BSTp of P4-P10 rats, labeled fibers
projecting from the BSTp traveled along two main routes, a
ventrolateral pathway that extended from the BSTp toward the medial
forebrain bundle and substantia innominata and a ventromedial pathway
that entered the medial preoptic area (Figs.
3, 4).
Comparable implants resulted in bundles of labeled fibers in both the
medial and lateral pathways, as well as labeled fibers in the MPNm. If
the DiI crystals were centered outside the borders of the BSTp, the
medial and lateral pathways and the terminal field in the MPNm were not
labeled. It is unlikely that variability in the placement of the DiI
crystal could account for the observed differences at each age, because our results were consistent within each group.
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Figure 3.
Preoptic projections of the BSTp.
AVPV, The distribution of DiI-labeled fibers (short
lines) at the level of the AVPV in male rats perfused on postnatal days
4 (P4), 7 (P7), 9 (P9), and 10 (P10) was
plotted onto a standard series of drawings derived from thionin-stained
sections through the preoptic region of a 10-d-old male rat.
Retrogradely labeled neurons have been omitted for clarity. The
dark gray region in the BST represents the area of DiI
diffusion from the implant site. 3V, Third ventricle;
aco, anterior commissure; adBST,
anterodorsal nucleus of the BST; ADP, anterodorsal
preoptic nucleus; avBST, anteroventral nucleus of the
BST; AVP, anteroventral preoptic nucleus;
AVPV, anteroventral periventricular nucleus;
CP, caudoputamen; LPO, lateral preoptic
area; LSv, lateral septal nucleus, ventral part;
LV, lateral ventricle; MaPO,
magnocellular preoptic nucleus; MePO, median preoptic
nucleus; MPO, medial preoptic area; MS,
medial septum; opt, optic tract; PS,
parastrial nucleus. See Figure 2 for additional abbreviations.
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Figure 4.
Preoptic projections of the BSTp.
MPN, Illustrations show the distribution of DiI-labeled
fibers at the level of the MPN in male rats perfused on
P4, P7, P9, and
P10. The dark gray area in the BST
represents the area of DiI diffusion immediately adjacent to the
implant site. BAC, Bed nucleus of the anterior
commissure; DBB, nucleus of the diagonal band;
GP, globus pallidus; MPNc, medial
preoptic nucleus, central part; MPNl, medial preoptic
nucleus, lateral part; MPNm, medial preoptic nucleus,
medial part; och, optic chiasm; PD,
posterodorsal preoptic nucleus; PvPo, preoptic
periventricular nucleus. See Figures 2 and 3 for additional
abbreviations.
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Labeled fibers projecting from the BSTp into the medial preoptic area
were sparse in P2 and P4 males. In P4 animals, the ventrolateral and
ventromedial pathways were evident, and labeled fibers extended beyond
the borders of the BST into the preoptic area but did not reach either
the MPNm or the AVPV (Figs. 3, 4, P4). In contrast to
the labeling pattern observed in P4 males, labeled fibers did not
extend beyond the morphological border of the BST and did not enter the
medial preoptic area in P2 males. In P7 males, labeled fibers from the
BSTp extended well into the medial preoptic area and were distributed
along the lateral border of the MPN but were sparsely distributed
within the MPNm (Figs. 3, 4, P7). The projections to
the AVPV and MPNm develop rapidly between P9 and P10. The pattern of
labeled fibers in the medial preoptic area of P9 males resembled that
seen in P10 males (see below). Although both the MPNm and AVPV
contained labeled fibers in P9 males, the density was much less than
that observed in these nuclei in P10 males, and the distribution was
much less uniform (Figs. 3, 4, P9). We did observe labeled
fibers projecting through the fornix in cases in which the DiI crystal
was placed immediately adjacent to the fornix but still within the
confines of the BSTp. We have represented this pattern of labeled
fibers in our illustrations; however, these fibers do not appear to
originate in the BSTp because they were not present in all cases.
In male rats perfused on P10, the pattern of projections from the BSTp
to the preoptic region resembled that of adult males with a dense
plexus of labeled fibers found in both the MPNm and AVPV (Figs. 3, 4,
P10). The density of labeled fibers in the MPNm was much
greater than was that in the MPNl (Fig. 4, P10). Similarly, a higher density of labeled fibers was observed in the AVPV than in
adjacent parts of the preoptic periventricular nucleus. The same mature
pattern of projections from the BSTp was also observed in P22 males.
Implants placed rostral, caudal, or ventral to the BSTp produced
distinctly different labeling patterns compared with that resulting
from implants centered in the BSTp. Implants centered lateral to the
BSTp did not label fibers in the medial preoptic area.
Similar DiI experiments performed in female rats perfused on P10
demonstrated the development of a sexual dimorphism in the projection
from the BSTp to the AVPV (Fig. 5).
Implant sites centered in the BSTp of three P10 females were obtained
that were comparable with those obtained in P10 males (Fig.
6). Crystalline deposits of DiI centered
within the morphological borders of the BSTp labeled fibers that passed
along the ventrolateral and ventromedial pathways. However, in contrast
to our findings in P10 males, the AVPV did not contain many labeled
fibers in females, although fibers were observed along the dorsal and
lateral borders of the AVPV (Figs. 5,
7C,D). The density
of labeled fibers in the MPNm of P10 females was also less than that
observed in the P10 male cases (Fig. 8), with the greatest density of labeled fibers along its dorsal and lateral borders (Fig. 8). A similar pattern of labeled fibers projecting from the BSTp was observed in P22 females. Analysis of
confocal images revealed a 20-fold difference in labeled fiber density
in the AVPV between P10 male and female rats (Fig.
9). Implants of DiI centered in the BSTp
of female rats perfused on P2 (n = 3), P4
(n = 1), or P6 (n = 3) also failed to
label projections from the BSTp into the AVPV or MPNm. In both male and
female cases, the MPNm contained labeled fibers only when the DiI
crystal was placed in the BSTp. This observation supports our
conclusion that the BSTp implants were comparable in male and
female rats. Moreover, the consistency of the results obtained in P10
males and P10 females suggests that it is unlikely that the observed
differences are attributable to differences in the placement of the DiI
crystals.
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Figure 5.
Sexually dimorphic projections of the BSTp.
Illustrations show the density and distribution of DiI-labeled fibers
projecting from the BSTp to the AVPV (A) and MPNm
(B) in female rats perfused on P10. The
dark gray region represents the area of DiI diffusion
from the implant site. See Figures 2-4 for abbreviations.
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Figure 6.
A, B, Low
magnification images of Nissl-stained sections showing the precise
placement of the DiI crystal in the BSTp of a P10 male
(A) and a P10 female (B)
rat. The arrows indicate the location of the center of
the DiI implant. C, D, Low magnification,
combined dark-field and fluorescence image montages showing the
appearance and distribution of DiI in comparable implants
obtained in P10 male (C) and female
(D) rats.
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Figure 7.
Confocal images of DiI-labeled fibers in the AVPV
of P10 male (A, C) and female
(B, D) rats. A,
B, A maximum projection image was derived from 10 confocal images collected (20× objective; NA, 0.75) through a total
distance of 20 µm. C, D, Ten confocal
images were collected (40× objective; NA, 1.3) through a total
distance of 10 µm to produce the projection image shown. Scale bars,
100 µm.
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Figure 8.
Confocal images of DiI-labeled fibers in the
medial preoptic nucleus of P10 male (A,
C) and female (B, D) rats.
A, B, A maximum projection image was
derived from six confocal images that were collected (20× objective;
NA, 0.75) through a total distance of 12 µm. C,
D, Ten images were collected (40× objective; NA, 1.3)
through a total distance of 10 µm. Scale bars, 100 µm.
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Figure 9.
Graphical representation of the sexual dimorphism
in the density of BSTp projections to the AVPV in P10 rats. The mean
density of labeled fibers represents the mean of the total numbers of
pixels in user-defined regions of thresholded, binarized images of
DiI-labeled fibers, derived from a maximum projection image of 10 confocal image planes and collected with a 40× objective from three
animals with comparable DiI implants centered in the BSTp.
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DISCUSSION |
The projection from the BSTp to the AVPV represents one of the
most robust morphological sex differences identified to date. The
demonstration of morphological or neurochemical sexual dimorphisms in
connectivity has usually depended on rigorous quantification, often at
the ultrastructural level (Raisman and Field, 1971; Matsumoto, 1991).
Clear exceptions to this general rule are some of the connections in
the avian vocal control system (Konishi and Akutagawa, 1985) and the
innervation of the dorsal tegmentum by laryngeal motor neurons in
Xenopus frogs (Kelley, 1996), but differences in the mammalian brain tend to be more subtle. The projection from the BSTp to
the AVPV is unique among hodological sex differences with respect to
its magnitude, suggesting that it may be a particularly useful
experimental model system for studying the development of sexually
dimorphic connections in the mammalian forebrain.
Sex steroid hormones can influence the numbers of neurons in sexually
dimorphic nuclei as well as the organization of their connections
(Arnold and Jordan, 1988; Simerly, 1995). Thus, sexually dimorphic
innervation of a target tissue may be controlled by regulating the
survival of neurons that project to a target region. For example, in
male zebra finches, neurons in the lateral magnocellular nucleus of the
anterior neostriatum send a strong projection to the robust nucleus of
the archistriatum. This projection is much weaker in females (Bottjer
and Arnold, 1997), and axonal transport studies suggest that selective
neuronal death of the neurons in the lateral magnocellular nucleus of
the anterior neostriatum that provide inputs to the robust nucleus
underlies the development of this sexually dimorphic pathway (Nordeen
et al., 1992). Similarly, testosterone is necessary for the survival of
neurons in the spinal nucleus of the bulbocavernosus, which innervates
the bulbocavernosus and levator ani muscles in the male rat. Nearly all
of these neurons die in the female, but their survival and the
maintenance of the innervation of the bulbocavernosus and levator ani
muscles can be rescued by neonatal administration of testosterone
(Breedlove, 1986). In both of these neural systems, development of a
sexually dimorphic connection appears to be dependent on testosterone
promoting the survival of a specific hormone-sensitive neuronal
population. The BSTp of male rats is larger than that of females and
appears to contain more neurons (del Abril et al., 1987), so it is not surprising to find stronger inputs to BSTp targets in the hypothalamus of male rats. However, simply on the basis of the greater size of the
BSTp, one would not predict the magnitude of the sexual dimorphism in
the density of the projections to the AVPV. The BSTp also provides a
stronger input to the MPNm in male rats than it does in females, but in
contrast to its extremely weak projection to the AVPV, the BSTp sends a
substantial input to the MPNm in females. Thus, the projection from the
BSTp to the AVPV appears to be particularly dimorphic, relative to the
other projections of the BSTp.
Numbers of projection neurons that survive into adulthood are often
influenced by the size of their targets (Breedlove, 1985; Sohal, 1992;
Thorn and Truman, 1994). Because the AVPV contains more neurons in
females, it might be reasonable to expect that it would receive a
stronger input from the BSTp in animals of this sex because greater
numbers of neurons in target regions tend to promote survival of
neurons that provide afferent connections. However, the projection from
the BSTp to the AVPV seems to be unique among sexually dimorphic
pathways in that a smaller population of cells in the AVPV of males is
innervated by a greater number of BSTp neurons, relative to the
proportions of neurons that comprise this connection in females. Thus,
the elevated levels of sex steroid hormones present in males during the
perinatal period appear to decrease the number of neurons in the AVPV,
while apparently promoting its innervation by neurons in the BSTp. This
observation is consistent with the idea that sex steroid hormones can
independently influence neuronal survival and the organization of
connectivity within the same neural pathway. The result of the
differential effect of sex steroids on the projection from the BSTp to
the AVPV seems to be a dramatic convergence of sensory information on
AVPV neurons in male rats.
The projection from the BSTp to the AVPV is established between
postnatal days 9 and 10 in male rats, and both the density and
distribution of labeled fibers are markedly different in females. By
P10, the density of BSTp fibers innervating the AVPV is ~20-fold greater in males, whereas in females the few fibers that are present are clustered along the periphery of the nucleus. The projection from
the BSTp to the MPNm develops a bit earlier than that to the AVPV.
Labeled fibers were observed in the MPNm as early as P7 in male rats,
but the adult pattern of innervation of the AVPV was not apparent until
P10. The projections from the BSTp to the MPNm and the AVPV both appear
to be relatively mature by P10.
For conceptual purposes the development of the projection from the BSTp
to the AVPV can be divided into two phases. First, axons leave the BSTp
and enter the medial preoptic area; this occurs in both sexes. Second,
axons preferentially innervate the MPNm and AVPV in male rats but
provide weaker inputs in females, suggesting that the specificity of
the sexual differentiation lies in the second phase. Axons passing
through the medial preoptic area appear to be attracted toward the MPNm
and AVPV with very few axons deviating from their medial course. This
type of directed axonal growth to target tissue is reminiscent of the
development of thalamocortical projections to layer IV of the visual
cortex (Agmon et al., 1993). Axons from the lateral geniculate nucleus enter layer IV of the visual cortex in bundles and terminate in specific target fields without sending collateral branches to inappropriate targets. In contrast, transient projections have been
observed in corticospinal pathways (O'Leary, 1992). Although it
remains possible that a transient connection between the BSTp and the
AVPV may occur in the female, this seems to be unlikely because we did
not observe substantial numbers of BSTp fibers in the AVPV of female
rats at any of the ages examined. If such a transient connection occurs
in females, it must be established and retracted between postnatal days
10 and 22. Our results suggest that very few axons from the BSTp ever
reach the AVPV in females, and those that do are distributed along the
lateral border of the nucleus, rather than being evenly dispersed
within the AVPV as they are in the male. Similarly the BSTp may
initially provide input to the AVPV and MPN in each sex, but the
projection to the AVPV may be selectively pruned back during the
perinatal period in females. This possibility also seems to be unlikely
because the projections from the BSTp to the AVPV and MPNm seem to
strengthen with postnatal age in both males and females, and a diffuse
arbor of labeled axons is not observed in the MPNm or AVPV until the fibers reach their targets. Nor did axons projecting toward the MPNm
and AVPV appear to be highly branched as they traversed the medial
preoptic area. Thus, the development of the projections from the BSTp
to the AVPV appears to be directed. Whether chemoattractive or
chemorepulsive factors or contact with extracellular guidance molecules
(Goodman, 1996) mediate the development of this sexually dimorphic
pathway from the BSTp to the AVPV remains to be determined.
The function of the sexually dimorphic connection from the BSTp to the
AVPV is not known, but presumably this pathway conveys olfactory
information to the AVPV. Thus, the sexually dimorphic organization of
afferent and efferent connections of the AVPV may contribute to
sex-specific patterns of convergence or divergence of sensory
information affecting neuroendocrine physiology. The exploration of
molecular mechanisms underlying the development of this pathway
represents a unique opportunity for gaining a greater understanding of
how hormones regulate the architecture of the sexually dimorphic neural
systems that regulate reproduction.
| |
FOOTNOTES |
Received Sept. 11, 1997; revised Jan. 30, 1998; accepted Feb. 4, 1998.
This work was supported by National Institutes of Health Grants
MH49236, RR00163, and HD18185. We thank Meigan Crabtree for expert
technical assistance and C. Houser for preparation of this manuscript.
We are also grateful to Dr. S. Amara of the Vollum Institute (Oregon
Health Sciences University) for use of the Bio-Rad MRC 1000 confocal
microscope.
Correspondence should be addressed to Dr. Richard B. Simerly, Program
in Neuroscience, Oregon Health Sciences University, 3181 Southwest Sam
Jackson Park Road, Portland, OR 97201.
| |
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Abstract
Introduction
