tmp1BC4 TMP

THE JOURNAL OF COMPARATIVE NEUROLOGY 494:190 214 (2006)
Comparative Study of the Sources of

Neuronal Projections to the Site of
Gonadotrophin-Releasing Hormone
Perikarya and to the Anteroventral
Periventricular Nucleus in Female Rats
JOEL D. HAHN AND CLIVE W. COEN*
School of Biomedical Sciences, Kings College London, London SE1 1UL, United Kingdom
ABSTRACT
The rat ovulatory cycle is dependent on the preoptic region encompassing the gonadotrophinreleasing hormone (GnRH) perikarya and the anteroventral periventricular nucleus (AVPV).
Retrograde tract tracing was used to identify and compare the sources of inputs to these sites in
female rats. Within the telencephalon and diencephalon, the incidence of retrograde labelling
from both sites was moderate to abundant in the ventral lateral septum, posteromedial bed
nucleus of the stria terminalis, amygdalohippocampal area and the periventricular, medial
preoptic, anterodorsal preoptic, dorsomedial suprachiasmatic, arcuate, and posterior ventrolateral ventromedial hypothalamic nuclei. In these regions, the incidence of retrograde labelling was
either greater from the AVPV than from the GnRH perikarya site or similar from both sites. In
the medial amygdaloid, parastrial, striohypothalamic, and ventral premammillary nuclei, the
retrograde labelling from the AVPV greatly exceeded the sparse incidence from the GnRH
perikarya site. In contrast, retrograde labelling from the GnRH perikarya site predominated in
the median preoptic, lateroanterior and dorsomedial hypothalamic nuclei, subparaventricular
zone, and retrochiasmatic area; it was abundant in the AVPV. Caudal to the diencephalon,
retrograde labelling from either site was sparse, except in the lateral parabrachial nucleus, which
displayed a particularly high incidence from the GnRH perikarya site. Other mesencephalic
regions labelled from either site included the periaqueductal gray and dorsal and median raphe
nuclei. The most caudal labelling was found in the ventrolateral medulla and region of the
solitary tract nucleus; this was almost exclusively from the GnRH perikarya site. These ndings
further elucidate the neuroanatomical connections underlying the control of the ovulatory cycle.
J. Comp. Neurol. 494:190 214, 2006. 2005 Wiley-Liss, Inc.
Indexing terms: GnRH; LHRH; bed nucleus of the stria terminalis; lateral parabrachial nucleus;
lateral septum; premammillary nucleus; preoptic area; ventromedial
hypothalamic nucleus; reproductive cycle
The neurons that synthesize gonadotrophin-releasing

hormone (GnRH) constitute the nal common pathway in
the central control of the ovulatory cycle. By direct or
indirect means, these neurons respond to changing concentrations of gonadal steroids during the estrous cycle
and regulate gonadotrophin release from the pituitary
gland. In their absence, such as in Kallmanns syndrome,
there is a loss of fertility (Schwanzel-Fukuda et al., 1989).
Just medial and caudal to their principal site in rats lies
the anteroventral periventricular nucleus (AVPV), a nucleus that lacks GnRH cell bodies but plays an essential
role in generating the preovulatory surge of luteinizing
hormone (LH; Wiegand et al., 1978, 1980; Terasawa et al.,
2005 WILEY-LISS, INC.
1980; Wiegand and Terasawa, 1982; Ronnekleiv and

Kelly, 1988).
Grant sponsor: Biotechnology and Biological Sciences Research Council

(studentship to J.D.H.); Grant sponsor: Wellcome Trust; Grant number:
060202.
*Correspondence to: Clive W. Coen, School of Biomedical Sciences,
Kings College London, London SE1 1UL, United Kingdom.
E-mail: clive.coen@kcl.ac.uk.
Received 22 March 2005; Revised 1 July 2005; Accepted 2 August 2005
DOI 10.1002/cne.20803
Published online in Wiley InterScience (www.interscience.wiley.com).
Abbreviations
2n
3V
4V
10
12
ac
aca
ACo
ADP
AHA
AHC
AHP
AHiAL
AHiPM
Amb
AP
Aq
ArcD
ArcL
ArcLP
ArcM
ArcMP
AVPV
BAOT
Bar
BMA
BSTMA
BSTMPI
BSTMPL
BSTMPM
BSTMV
cc
CeM
CGPn
CLi
CM
cp
CVL
D3V
DA
DG
DLPAG
DMHd
DMHv
DR
DRC
DRD
DRV
DRVL
DRVL
DTM
EW
f
fr
HDB
ic
IPC
IPR
LA
LC
lfp
LH
LHp
LPAG
LPBC
LPBD
LPBE
LPBS
LPBV
LPO
LRt
LSI
LSV
LV
me5
MeAD
MePD
optic nerve
3rd ventricle
4th ventricle
dorsal motor nucleus of the vagus
hypoglossal nucleus
anterior commissure
anterior commissure, anterior part
anterior cortical amygdaloid nucleus
anterodorsal preoptic nucleus
anterior hypothalamic area, anterior part
anterior hypothalamic area, central part
anterior hypothalamic area, posterior
amygdalohippocampal area, anterolateral part
amygdalohippocampal area, posteromedial part
ambiguus nucleus
area postrema
aqueduct (Sylvius)
arcuate nucleus, dorsal part
arcuate nucleus, lateral part
arcuate hypothalamic nucleus, lateroposterior part
arcuate nucleus, medial part
arcuate hypothalamic nucleus, medial posterior part
anteroventral periventricular nucleus
bed nucleus of the accessory olfactory tract
Barringtons nucleus
basomedial amygdaloid nucleus, anterior part
bed nucleus of the stria terminalis, medial division, anterior part
bed nucleus of the stria terminalis, medial division, posterointermediate part
bed nucleus stria terminalis, medial division, posterolateral part
bed nucleus of the stria terminalis, medial division, posteromedial part
bed nucleus of the stria terminalis, medial division, ventral
corpus callosum
central amygdaloid nucleus, medial division
central gray of the pons
caudal linear nucleus of the raphe
central medial thalamic nucleus
cerebral peduncle, basal part
caudoventrolateral reticular nucleus
dorsal 3rd ventricle
dorsal hypothalamic area
dentate gyrus
dorsolateral periaqueductal gray
dorsomedial hypothalamic nucleus, dorsal part
dorsomedial hypothalamic nucleus, ventral part
dorsal raphe nucleus
dorsal raphe nucleus, caudal part
dorsal raphe nucleus, dorsal part
dorsal raphe nucleus, ventral part
dorsal raphe nucleus, ventrolateral part
dorsal tuberomammillary nucleus
dorsal tuberomammillary nucleus
Edinger-Westphal nucleus
fornix
mbria of the hippocampus
fasciculus retroexus
nucleus of the horizontal limb of the diagonal band
internal capsule
interpeduncular nucleus, caudal subnucleus
interpeduncular nucleus, rostral subnucleus
lateroanterior hypothalamic nucleus
locus coeruleus
longitudinal fasciculus of the pons
lateral hypothalamic area
lateral hypothalamus, posterior
lateral periaqueductal gray
lateral parabrachial nucleus, central part
lateral parabrachial nucleus, dorsal part
lateral parabrachial nucleus, external part
lateral parabrachial nucleus, superior part
lateral parabrachial nucleus, ventral part
lateral preoptic area
lateral reticular nucleus
lateral septal nucleus, intermediate part
lateral septal nucleus, ventral part
lateral ventricle
mesencephalic trigeminal tract
medial amygdaloid nucleus, anterodorsal part
medial amygdaloid nucleus, posterodorsal part
MePV
mfb
ml
MM
MnPO
MnR
mp
MPA
MPB
MPOL
MPOM
MRe
MS
mt
MTu
opt
ox
PaAP
PAG
PaMP
PaPo
PaV
pc
Pe
PH
PiRe
PL
pm
PMD
PMV
Pn
pv
PS
PVA
PVN
PVP
RCh
Re
RelC
RVL
SCN
SCNc
SCNdl
SCNdm
SCNvl
SCNvm
scp
SFO
sm
SO
sol
SolC
SolDM
SolM
sox
SPZ
st
StA
StHy
SubCA
SubI
SuM
SuMM
TC
tfp
TS
vhc
VLL
VLPAG
VMHA
VMHDM
VMHVL
VMHVLp
VMPO
VTM
xscp
ZI
ZID
ZIV
medial amygdaloid nucleus, posteroventral part

medial forebrain bundle
medial lemniscus
medial mammillary nucleus, medial part
median preoptic nucleus
median raphe nucleus
mammillary peduncle
medial preoptic area
medial parabrachial nucleus
medial preoptic nucleus, lateral part
medial preoptic nucleus, medial part
mammillary recess of the 3rd ventricle
medial septal nucleus
mammillothalamic tract
medial tuberal nucleus
optic tract
optic chiasm
paraventricular hypothalamic nucleus, anterior parvicellular part
periaqueductal gray
paraventricular hypothalamic nucleus, medial parvicellular part
paraventricular hypothalamic nucleus, posterior part
paraventricular hypothalamic nucleus, ventral part
posterior commissure
periventricular hypothalamic nucleus
posterior hypothalamic area
pineal recess
paralemniscal nucleus
principal mammillary tract
premammillary nucleus, dorsal part
premammillary nucleus, ventral part
pontine nuclei
periventricular ber system
parastrial nucleus
paraventricular thalamic nucleus, anterior part
paraventricular hypothalamic nucleus
paraventricular thalamic nucleus, posterior part
retrochiasmatic area
reuniens thalamic nucleus
recess of the inferior colliculus
rostroventrolateral reticular nucleus
suprachiasmatic nucleus hypothalamus
suprachiasmatic nucleus (central part)
suprachiasmatic nucleus, dorsolateral
suprachiasmatic nucleus, dorsomedial
suprachiasmatic nucleus, ventrolateral
suprachiasmatic nucleus, ventromedial
superior cerebellar peduncle (brachium conjunctivum)
subfornical organ
stria medullaris of the thalamus
supraoptic nucleus
solitary tract
nucleus of the solitary tract, commissural part
nucleus of the solitary tract, dorsomedial part
nucleus of the solitary tract, medial part
supraoptic decussation
subparaventricular zone
stria terminalis
strial part of the preoptic area
striohypothalamic nucleus
subcoeruleus nucleus, alpha part
subincertal nucleus
supramammillary nucleus
supramammillary nucleus, medial part
tuber cinereum area
transverse bers of the pons
triangular septal nucleus
ventral hippocampal commissure
ventral nucleus of the lateral lemniscus
ventrolateral periaqueductal gray
ventromedial hypothalamic nucleus, anterior part
ventromedial hypothalamic nucleus, dorsomedial part
ventromedial hypothalamic nucleus, ventrolateral part
ventromedial hypothalamic nucleus, posterior ventrolateral part
ventromedial preoptic nucleus
ventral tuberomammillary nucleus
decussation of the superior cerebellar peduncle
zona incerta
zona incerta, dorsal part
zona incerta, ventral part
192
Variation in ovarian steroid secretion is a fundamental
characteristic of the estrous cycle. In recent years, it has
been demonstrated that GnRH neurons express the beta
form of the nuclear estrogen receptor (ER), and may
consequently be subject to direct regulation by this steroid
(Kallo et al., 2001; Herbison and Pape, 2001; Hrabovszky
et al., 2001). Nevertheless, extensive data show that estrogen receptors, ER and ER, are widely distributed
throughout the brain (Simerly et al., 1990; Shughrue et
al., 1997; Li et al., 1997; Laamme et al., 1998; Zhang et
al., 2002) and that GnRH neurons are inuenced by a
neurochemically and functionally diverse neuronal network, which is sensitive to gonadal steroids (Levine et al.,
1991; Kalra, 1993; Petersen et al., 2003).
Among an estimated total of 100 million neurons in the
rat brain (Swanson, 1995), the number of GnRH neurons
ranges from about 1,200 to 1,500 (Witkin et al., 1982;
Merchenthaler et al., 1984; Wray and Hoffman, 1986);
their cell bodies are located principally in a region slightly
lateral and dorsal to the vascular organ of the lamina
terminalis (OVLT), extending caudolaterally (Witkin et
al., 1982; Merchenthaler et al., 1984; Wray and Hoffman,
1986; Coen et al., 1990). These neurons show increased
activity during the proestrous LH surge (Lee et al., 1990;
Hoffman et al., 1993; Porkka-Heiskanen et al., 1994; Petersen et al., 1995). The adjacent AVPV contains neurons
that also play a crucial role in the regulation of the rat
reproductive cycle. This sexually dimorphic nucleus [its
volume and cell density are greater in female rats (Bleier
et al., 1982)] is medial and caudal to the majority of GnRH
perikarya. From the rostral tip of the third ventricle,
immediately behind the OVLT, the AVPV extends caudally, tapering to end at the level of the rostral medial
preoptic nucleus, where the rest of the periventricular
nucleus continues.
In female rats, electrolytic lesions largely restricted to
the AVPV result in a loss of estrous cycles (Wiegand et al.,
1978, 1980; Wiegand and Terasawa, 1982; Ronnekleiv and
Kelly, 1988) and block LH surge induction by exogenous
estrogen followed by progesterone (Wiegand et al., 1978,
1980; Wiegand and Terasawa, 1982). Administration of an
antiestrogen to the AVPV disrupts the estrous cycle
(Orikasa and Sakuma, 2003). Tract tracing studies have
revealed projections from the AVPV to the vicinity of the
GnRH perikarya (Gu and Simerly, 1997); approximately
half of the AVPV cells forming these projections are immunoreactive for ER (Simonian et al., 1999). There is
also a dense concentration of ER-containing cells in the
AVPV (Kallo et al., 2001); this population is more numerous in female rats than in male rats (Orikasa et al., 2002).
At the time of the preovulatory LH surge, the immediate
early gene c-fos is expressed concurrently in the AVPV,
particularly in its medial region caudal to the OVLT, and
in GnRH cells (Le et al., 1999). There is a parallel decline
in LH surge-associated FOS expression in these two sites
in aged rats (Le et al., 2001).
Several studies on rats have described the origins of
forebrain inputs to the medial preoptic area, a site that is
caudal to the location of most GnRH perikarya (Sakumoto
et al., 1978; Day et al., 1980; Berk and Finkelstein, 1981;
Kita and Oomura, 1982; Chiba and Murata, 1985; Simerly
and Swanson, 1986; Leanza et al., 1989, 1991). Few previous retrograde tracing studies have focused on the rostral medial preoptic area, and none has comprehensively
described and compared the inputs to the site of GnRH
J.D. HAHN AND C.W. COEN

perikarya and to the AVPV. One study (employing uorescent microspheres as a tracer) has outlined the major
sources of forebrain and brainstem inputs to the rostral
medial preoptic area in rats (Simonian et al., 1999); two
comparable studies (both employing the retrograde tracer
Fluorogold) have been undertaken in sheep (Tillet, 1992;
Tillet et al., 1993). An additional two studies have identied origins of brainstem inputs to the vicinity of GnRH
perikarya in rats (Castaneyra-Perdomo et al., 1992;
Wright and Jennes, 1993); these employed horseradish
peroxidase (Castaneyra-Perdomo et al., 1992) or Fluorogold (Wright and Jennes, 1993).
In summary, the GnRH neurons and the AVPV are
essential for the central control of the ovulatory cycle. The
present study was designed to provide a detailed description and comparison of inputs to the principal site of
GnRH perikarya and to the AVPV; for this purpose, a
particularly sensitive tract tracing method involving cholera toxin B subunit (CTB) was employed.
MATERIALS AND METHODS

Animals
All procedures were licensed and approved under the
Animals (Scientic Procedures) Act of 1986 (United Kingdom). Studies were carried out on 41 female Wistar rats
weighing 220 350 g (Harlan Sera Lab, Loughborough,
United Kingdom); these were maintained in a controlled
environment (lights on at 0700 hours for 12 hours; ambient temperature of 21 1C; ad libitum access to food
and water).
Retrograde tracing
To enhance GnRH immunoreactivity, the rats were bilaterally ovariectomized under inhalation anesthesia
(Halothane; AstraZeneca, London, United Kingdom) at
least 10 days prior to tracer injection. Micropipettes for
tracer injection were made from glass capillaries (0.58 mm
i.d., 1.0 mm o.d., length 100 mm; Harvard Apparatus,
Edenbridge, United Kingdom). These were cleaned in 70%
ethanol and allowed to dry before being immersed in silicone uid (VWR International, Poole, United Kingdom)
and air dried. The micropipettes were produced using a
vertical electrode puller (Sutter Instrument Company, Novato, CA). A microforge (Narishige International, London,
United Kingdom) was used to burnish the tips; those
selected for use had an inner diameter of approximately
1520 m. The tracer CTB (0.5 mg/ml in 0.01 M sodium
phosphate buffer, pH 7.5; List Biological Laboratories
Inc., Campbell, CA) was drawn into the micropipettes
under suction; the outer barrel was wiped lightly toward
the tip with a moistened tissue to remove external tracer.
The tracer (approximately 1525 nl) was delivered by
Picospritzer pressure injection (General Valve, Faireld,
NJ). Coordinates for injection were determined with reference to an atlas of the rat brain (Paxinos and Watson,
1998). Because of slight individual variation between animals, these coordinates were found to be appropriate for
obtaining a tracer deposit including either the principal
site of the GnRH perikarya or the AVPV. Rats were anesthetized by intraperitoneal (ip) injection of ketamine hydrochloride (90 mg/kg; Pzer, Sandwich, United Kingdom)
and xylazine hydrochloride (10 mg/kg; Bayer, Bury St.
Edmunds, United Kingdom) and placed in a stereotaxic
RETROGRADE TRACING FROM GnRH CELL SITE OR AVPV

frame. Coordinates for Bregma were recorded, and the
skull was then drilled to produce a window for entry of the
micropipette. The rostrocaudal coordinate was 0.1 mm
behind Bregma. The lateral coordinate was 0.2 mm to the
left of the superior sagittal sinus, and the ventral coordinate was 7.8 mm below the surface of the brain. After the
micropipette had been lowered to the selected coordinates,
the meniscus of the tracer solution was monitored, by
using an operating microscope, while a pulse of nitrogen
gas (4 msec at 60 PSI) was delivered via the Picospritzer;
when the pulse resulted in movement of the meniscus, the
micropipette was left in situ for 10 minutes and then
retracted slowly. To enhance visualization of GnRH cells
bodies, rats were reanesthetized 2 4 weeks later with
ketamine and xylazine (ip) and placed in a stereotaxic
frame. A 10-l injection of colchicine (10 g/l; SigmaAldrich, London, United Kingdom) was delivered into the
lateral ventricle with a 10-l microsyringe (Hamilton GB,
Carnforth, United Kingdom). On the following day, the
rats were deeply anesthetized and perfused transcardially
with 50 ml phosphate-buffered saline (PBS), pH 7.4, followed by 200 ml of 4% paraformaldehyde in PBS.
Tissue preparation
After perfusion, the brains were removed and postxed
for 48 hours in fresh xative. Fixed brains were blocked
and embedded in an agar support medium. Coronal sections (40 m) were cut with a Vibratome (Pelco International, Redding, CA). During collection, the sections were
allocated into four or six sequential sets. For long-term
storage, sections were transferred to an antifreeze solution (Watson et al., 1986) and maintained at 20C.
Immunocytochemistry
All immunocytochemistry (ICC) was performed on freeoating sections. Initially, dual ICC was performed on
sections encompassing the region of the GnRH cell bodies
to reveal the site and extent of the tracer deposit together
with the GnRH cell bodies. The tissue sections were exposed to 0.5% Triton X-100 in PBS for 45 minutes and
washed in PBS (at least six changes). They were then
treated with 1.0% sodium borohydride for 30 minutes and
washed in PBS (at least six changes). After exposure to
0.3% hydrogen peroxide in PBS for 15 minutes, they were
washed in PBS (at least three changes) and incubated in
2.0% donkey serum (Sigma-Aldrich) for 10 minutes, before
being washed in PBS for 2 minutes (one change). After
these pretreatments, sections were incubated in rabbit
anti-GnRH serum (1:20,000; Diasorin, Wokingham,
United Kingdom) for 48 hours at 4C. They were then
removed from the antibody solution and washed in PBS
for 2 hours (six changes), before incubation in biotinylated
donkey anti-rabbit IgG serum (1:1,500; Jackson Immunoresearch, West Grove, PA) for 2 hours at room temperature. After being washed in PBS for 45 minutes (six
changes), sections were incubated in an avidinbiotinylated horseradish peroxidase complex (ABC reagent, 1:1,000 in PBS; Vector, Peterborough, United Kingdom) for 90 minutes at room temperature. They were then
washed in PBS for 15 minutes (at least three changes) and
in Tris buffer, pH 7.6 (Sigma-Aldrich), for 15 minutes (at
least three changes). Sections were transferred to a glass
petri dish containing 25 ml Tris buffer with 0.003% hydrogen peroxide, 0.002% 3,3-diaminobenzidine tetrahydrochloride (DAB), and 1 mM ammonium nickel (II) sul-
193
fate hexahydrate. The reaction was monitored under a

microscope. When a sufcient strength of signal was obtained, the reaction was stopped by washing the sections
in Tris (two changes). The sections were then washed in
PBS (three changes) and incubated in 2% donkey serum
for 10 minutes. They were then washed in PBS for 2
minutes (one change) and incubated in goat anti-CTB
serum (1:30,000; List Biological Labs) for 48 hours at 4C.
Subsequently, the sections were treated according to the
procedures applied after the rst primary antiserum, except that they were incubated in biotinylated donkey antigoat IgG serum (1:1,500; Jackson Immunoresearch) and
ammonium nickel (II) sulfate hexahydrate was omitted
from the reaction solution. After the second immunocytochemical reaction, sections were washed in Tris and
mounted from Elvanol (0.3% polyvinyl alcohol; SigmaAldrich) onto glass slides. The mounted sections were
allowed to air dry for several days before being passed
through an ascending series of ethanol solutions (50%,
70%, 95%, and 100%) and then xylene; glass slips were
then secured on the slides with DPX mountant. After the
dual ICC for the tracer injection site and GnRH cells,
single ICC was performed on a complete set of sections to
reveal the location of the retrogradely labelled CTBcontaining cell bodies. This ICC was performed according
the method already described for the detection of CTB,
except that ammonium nickel (II) sulfate hexahydrate
was added to the reaction solution. In addition, in some
cases, a Nissl counterstain (0.5% thionin in pH 4.0 acetate
buffer) was applied to the slide-mounted sections in order
to discern the cytoarchitecture.
Control procedures for ICC

Omission of the primary antiserum (for CTB or GnRH),
with all other processing steps remaining unchanged, resulted in a complete absence of immunoreactive proles.
Immunoreactivity was also absent when the immunocytochemical procedures for visualizing CTB were applied to
brains that had not been treated with the tracer. Increasing dilutions of the primary antisera led to a commensurate attenuation of the immunoreactive signal. No immunoreactivity was observed when the GnRH antiserum had
been pretreated with GnRH (5 g/ml).
Analysis
Analysis was performed with a microscope equipped
with Plan-NeoFluar semiapochromatic objectives (Axioskop 2 Plus; Carl Zeiss Ltd., Welwyn Garden City, United
Kingdom). Quantication of retrograde labelling was established with a reticule. The mean number of labelled
cells in 200 200 m areas was determined (taking up to
eight measurements per neuroanatomical region), and
this result was allocated into one of seven categories for
each region: 0 (), 15 (), 6 10 (), 1120 (),
2135 (), 36 60 (), and 60 ().
The category to which the area belongs was tabulated and
represented graphically on rat brain atlas diagrams, using
markers to represent each of the seven levels of abundance. Photomicrographs were obtained with a digital
camera (AxioCam; Carl Zeiss Ltd.). To obtain nal images
as true to the microscopic view as possible, and to achieve
optimal presentation, the digitally acquired images were
reoriented and/or adjusted for brightness, contrast, or
color balance, either at the time of acquisition or subse-
194
quently in photoimaging software (Adobe Photoshop

v5.02).
RESULTS
The location and extent of the cholera toxin tracer deposit in each animal varied; the sites ranged (rostrocaudally) from the horizontal limb of the diagonal band to the
medial preoptic nucleus. To provide a clear comparison
between inputs to the principal site of GnRH perikarya
and to the AVPV, our most detailed analysis focuses on
cases in which the tracer deposit showed minimal spread
from either of these sites (Fig. 1a,b). Data are also provided from deposits that included these sites but were not
restricted to them (Fig. 1d g). Any case in which the
microinjection appeared to have ruptured the ependymal
lining of the third ventricle was rejected. Of the 41 cases
examined, 6 of the tracer deposits included the site of
GnRH perikarya and 10 included the region of the AVPV
that shows elevated FOS expression at the time of the LH
surge (Le et al., 1999, 2001). The retrograde labelling
identied in each of these cases was consistent with the
pattern observed in the cases showing minimal tracer
spread from the site of GnRH perikarya or the AVPV. The
results are summarized in Table 1 and Figures 2 and 3. In
the diencephalon and subcortical telencephalon, the great
majority (about 90%) of retrogradely labelled cells were
located ipsilateral to the site of tracer deposit; caudal to
the diencephalon, this gure dropped to about 80%. In
both rostral and caudal regions, the cells that were contralateral to the tracer deposit displayed a distribution
similar to that of the ipsilateral cells.
Telencephalon
Retrograde labelling was not present within any region
of the neocortex and was found exclusively in subcortical
regions of the telencephalon.
Septum, bed nucleus of the stria terminalis, and subfornical organ. Within the lateral septum, numerous
cells were retrogradely labelled from the AVPV; these
were more abundant than those retrogradely labelled
from the site of GnRH perikarya. From either site, labelled cells were particularly abundant within the ventral
subdivision of the lateral septal nucleus (Fig. 4a,b); elsewhere in the septum, labelled cells were relatively few and
isolated. In the medial division of the bed nucleus of the
stria terminalis (BST), retrograde labelling was generated
from the site of both GnRH perikarya and the AVPV (Fig.
4c,d); this was particularly abundant in the posteromedial
subdivision, where retrograde labelling from the AVPV
predominated (Fig. 4d). A low incidence of retrograde labelling was found in the subfornical organ from either the
GnRH perikarya site or the AVPV.
Amygdaloid region. From the AVPV, a moderate incidence of labelling was found in the posterodorsal subdivision of the medial amygdaloid nucleus (Fig. 4j); fewer
labelled cells were produced in this region from the site of
GnRH perikarya (Fig. 4i). From both sites, a higher incidence of back-labelling was found in the amygdalohippocampal area, the incidence from the AVPV being particularly prominent (Fig. 4o,p); in contrast, only isolated cells
were labelled in the basomedial and central nuclei of the
amygdala.
Fig. 1. a,b: Brighteld photomicrographs showing two immunocytochemically identied deposits of cholera toxin (CT) tracer (dark gray
area), one (a; #22) encompassing gonadotrophin-releasing hormone
(GnRH)-immunoreactive (IR) perikarya (black proles), the other (b;
#12) located in the region of the anteroventral periventricular nucleus
(AVPV). c: Brighteld photomicrograph showing perikarya within the
AVPV that have been retrogradely labelled with CT (black proles)
from the tracer site encompassing GnRH-IR perikarya (a; #22). Note
the absence of GnRH-IR perikarya within the AVPV (a c). d g:
Brighteld photomicrographs showing two additional CT deposits,
one (d,e; #30) including the AVPV, in addition to the site of GnRH-IR
perikarya (lateral to the AVPV), the other (f,g; #11) including the
AVPV (f) and extending caudally into the medial preoptic nucleus
(MPO; g). Scale bars 100 m in a,d g; 50 m in b,c.
195
TABLE 1. Summary of the Sources of Neuronal Projections to Regions Including the Site of Gonadotrophin Releasing Hormone Cell Bodies
or the Anteroventral Periventricular Nucleus1
Anatomical Region
Telencephalon
Nucleus of the horizontal limb of the diagonal band
Lateral septal n., intermediate
Lateral septal n., ventral
Bed nucleus stria terminalis, medial division, ventral
Bed nucleus stria terminalis, medial division, anterior
Bed nucleus stria terminalis, med. div., posteromedial
Bed nucleus stria terminalis, med. div., posterointermediate
Medial amygdaloid nucleus, anterodorsal
Medial amygdaloid nucleus, posterodorsal
Medial amygdaloid nucleus, posteroventral
Basomedial amygdaloid nucleus, anterior
Central amygdaloid nucleus, medial division
Amygdalohippocampal area, anterolateral
Amygdalohippocampal area, posteromedial
Diencephalon (and adjacent rostral regions)
Anteroventricular periventricular nucleus
Periventricular hypothalamic nucleus
Medial preoptic area
Medial preoptic nucleus, medial
Medial preoptic nucleus, lateral
Median preoptic nucleus
Subfornical organ
Anterodorsal preoptic nucleus
Parastrial nucleus
Lateral preoptic area
Striohypothalamic nucleus
Suprachiasmatic nucleus, ventromedial
Suprachiasmatic nucleus, ventrolateral
Suprachiasmatic nucleus (central part)
Suprachiasmatic nucleus, dorsomedial
Suprachiasmatic nucleus, dorsolateral
Subparaventricular zone (dorsal to suprachiasmatic nucleus)
Lateroanterior hypothalamic nucleus
Anterior hypothalamic area, anterior
Anterior hypothalamic area, central
Anterior hypothalamic area, posterior
Lateral hypothalamic area
Retrochiasmatic area
Paraventricular hypothalamic nucleus, anterior parvocellular
Paraventricular hypothalamic nucleus, medial parvocellular
Paraventricular hypothalamic nucleus, ventral
Paraventricular thalamic nucleus, anterior
Zona incerta
Arcuate nucleus, dorsal
Arcuate nucleus, lateral
Arcuate nucleus, medial
Arcuate nucleus, lateral posterior
Arcuate nucleus, medial posterior
Ventromedial hypothalamic nucleus, anterior
Ventromedial hypothalamic nucleus, dorsomedial
Ventromedial hypothalamic nucleus, posterior ventrolateral
Dorsomedial hypothalamic nucleus, dorsal
Dorsomedial hypothalamic nucleus, ventral
Dorsal hypothalamic area
Lateral hypothalamus, posterior
Posterior hypothalamic area
Dorsal tuberomammillary nucleus
Premammillary nucleus, dorsal
Premammillary nucleus, ventral
Supramammillary nucleus, medial
Mesencephalon and Rhombencephalon
Ventrolateral periaqueductal gray
Dorsal raphe nucleus
Caudal linear nucleus of the raphe
Median raphe nucleus
Locus coeruleus
Barringtons nucleus
Lateral parabrachial nucleus, central
Lateral parabrachial nucleus, dorsal
Lateral parabrachial nucleus, external
Lateral parabrachial nucleus, superior
Lateral parabrachial nucleus, ventral
Central gray of the pons
Caudoventrolateral reticular nucleus
Lateral reticular nucleus
Ventrolateral medulla
Nucleus of the solitary tract, medial
Nucleus of the solitary tract, commissural
1
GnRH #22
AVPV #12
GnRH AVPV
#30
AVPV MPO
#11
Sites of retrograde labelling from a deposit of cholera toxin tracer located either (#22) in the immediate vicinity of gonadotrophin releasing hormone (GnRH) perikarya, (#12)
predominantly within the anteroventral periventricular nucleus (AVPV), (#30) in the AVPV but also encompassing the site of GnRH perikarya, lateral to the AVPV, or (#11) in
the AVPV but also extending caudally into the medial preoptic nucleus (MPO). Ratings reect the abundance of perikarya immunoreactive for the tracer per 200200 m area
at a given site: , 15; , 6 10; , 1120; , 2135; , 36 60; , 60. The nomenclature is according to Paxinos & Watson (1998) with minor
modications.
196
Diencephalon
Preoptic region. There were numerous retrogradely
labelled cells in the AVPV from the site of GnRH
perikarya (Fig. 1a,c). Retrograde labelling was also abundant from the AVPV or the GnRH perikarya site in the
periventricular, parastrial, striohypothalamic, and medial
subdivision of the medial preoptic nuclei; in these regions,
labelling from the AVPV generally predominated, most
notably in the parastrial and striohypothalamic nuclei.
Within the medial subdivision of the medial preoptic nucleus, cells retrogradely labelled from the AVPV were
slightly more numerous than from the site of GnRH
perikarya; within the lateral subdivision of this nucleus
there was a lower number of labelled cells, but those from
the GnRH perikarya site predominated. A moderate incidence of retrograde labelling was found in the anterodorsal preoptic nucleus from either site and in the median
preoptic nucleus from the site of GnRH perikarya; the
incidence of labelling from the AVPV in the median preoptic nucleus was relatively low. Elsewhere in the preoptic
area, labelling was sparse.
Suprachiasmatic nucleus region. Retrograde labelling was found within the suprachiasmatic nucleus (SCN)
from the site of GnRH perikarya and from the AVPV (Fig.
4e,f). Labelling from either site was at a moderately low
incidence in the dorsomedial subdivision of this nucleus; it
was also present in the ventromedial region, where cells
retrogradely labelled from the site of GnRH perikarya
were less numerous. Sparse labelling was also seen in the
dorsolateral region from either site. In the ventrolateral
and central regions of the SCN, labelling was very rare
unless the tracer deposit included the medial preoptic
nucleus caudal to the site of GnRH perikarya and the
AVPV. Moderate retrograde labelling from the GnRH
perikarya site was present in the subparaventricular zone
immediately dorsal to the SCN and in the laterally adjacent lateroanterior hypothalamic nucleus; at both of these
sites, the retrograde labelling from the AVPV was only
sparse. Moderate numbers of retrogradely labelled cells
were present in the retrochiasmatic area, where labelling
from the GnRH perikarya site predominated.
Hypothalamic and thalamic paraventricular nuclei.
The incidence of retrogradely labelled cells in the hypothalamic paraventricular nucleus (PVN) from either the
site of GnRH perikarya or the AVPV was generally sparse,
but labelled cells were slightly more numerous in its anterior subdivision; isolated cells labelled from either site
were also found in the zone of the lateral hypothalamus
immediately lateral to the PVN. It should be noted that
neurons expressing corticotrophin-releasing hormone
(CRH) are abundant in the medial parvocellular subdivision of the PVN, where retrograde labelling was rare.
Sparse labelling was also found in the anterior division of
the thalamic paraventricular nucleus from both sites.
Arcuate, ventromedial, and dorsomedial nuclei. A
predominantly moderate level of retrograde labelling was
found in the arcuate nucleus, encompassing its full extent,
from the site of the GnRH perikarya or the AVPV (Fig.
4g,h,m,n). The highest level of retrograde labelling from
the AVPV in the entire brain was present in the posterior
subdivision of the ventrolateral division of the ventromedial nucleus (VMHVLp; Fig. 4l); a smaller number of labelled cells was found in this region from the site of the
GnRH perikarya (Fig. 4k). The rest of the VMH was only
sparsely labelled from either site. Retrograde labelling of

the dorsomedial nucleus (DMH) was produced from both
sites, the incidence being more substantial in the ventral
region; in both dorsal and ventral regions, the labelling
from the site of GnRH perikarya predominated. The central subdivision of the DMH was only sparsely labelled
from either site.
Caudal diencephalon. Abundant retrograde labelling was found in the ventral premammillary nucleus
from the AVPV (Fig. 4n); very little was found in this
nucleus from the GnRH perikarya site (Fig. 4m). A moderate amount of labelling was present in the medial supramammillary nucleus from the AVPV, with less from
the GnRH perikarya site. Retrograde labelling in the rest
of the caudal diencephalon, such as the posterior hypothalamic area, dorsal premammillary nucleus, and dorsal
tuberomammillary nucleus was at a relatively low incidence from both sites.
Mesencephalon and rhombencephalon

In contrast to the generally higher abundance of retrograde labelling from the AVPV within the diencephalon,
retrograde labelling from the site of GnRH perikarya predominated in brain regions caudal to the diencephalon.
Within the lateral parabrachial nucleus, an abundance of
cells retrogradely labelled from the site of GnRH
perikarya was found in its central subdivision (Fig. 4q). A
moderate incidence of such cells was seen in the dorsal
and superior lateral parabrachial nucleus. In this region,
relatively few cells were labelled from the AVPV (Fig. 4r).
Few labelled cells were found in the locus coeruleus, and
these were present only in cases in which the injection site
encompassed part of the medial preoptic nucleus caudal to
the GnRH perikarya and AVPV. These labelled cells were
relatively small; in Nissl-counterstained tissue, they did
not correspond to the intensely stained cells, with large
cell bodies, characteristic of the local noradrenaline neurons.
Retrograde labelling from the AVPV was not observed
caudal to the central gray of the pons, where only isolated cells were found. A few cells retrogradely labelled
from the AVPV or GnRH perikarya site were found in
the following mesencephalic regions: ventrolateral periaqueductal gray, dorsal raphe nucleus, caudal linear
nucleus of the raphe, median raphe nucleus, and Barringtons nucleus. More caudally, a few isolated cells
from the site of GnRH perikarya were found in the
following regions of the rhombencephalon: ventrolateral
medulla (C1/A1 region), lateral reticular nucleus, and
nucleus of the solitary tract.
DISCUSSION
The present study describes and compares retrograde
labelling from two regions in the preoptic area: the principal site of GnRH perikarya and the AVPV. Each of these
regions is essential for maintenance of the rat ovulatory
cycle. To place these neuroanatomical ndings within a
physiologically relevant context, the sites displaying retrograde labelling will be discussed with reference to functional studies, involving local stimulation or ablation, and
to the local presence of estrogen receptors.
Inferences from tract tracing procedures should be qualied by anatomical and technical considerations. The preoptic area is implicated in numerous functions apart from

those directly concerned with reproduction; these include
the regulation of body temperature (Szymusiak and Satinoff, 1984), sleep (McGinty and Sterman, 1968), blood
pressure and uid and electrolyte balance (Brody and
Johnson, 1980). Additional functional roles for this area
cannot be excluded. Thus, there are no a priori reasons for
assuming that the neurons retrogradely labelled in this
study are necessarily involved in the regulation of reproduction. Because the sites targeted by the tracer contain
heterogeneous cells, it will be important to complement
the present ndings with anterograde tracing studies at
confocal and electronmicroscopic levels; it will also be necessary to phenotype the afferents. Furthermore, it should
be recognized that neuronal inputs to the relatively isolated GnRH perikarya found lateral and/or caudal to their
principal site may differ from those reported here.
The region designated as the principal site of GnRH
perikarya is one in which only a small proportion of the
local neurons expresses GnRH. There is considerable speculation about GnRH regulation by non-GnRH neurons in
their vicinity (Herbison, 1998), but such neurons remain
largely uncharted. A recent study (Campbell et al., 2005)
revealed that GnRH neurons in this region have exceptionally long dendrites in mice; their distal processes cannot be detected by GnRH immunoreactivity. The predominantly dorsoventral orientation of such dendrites
(Campbell et al., 2005) suggests that the tracer deposits in
the present study would have encompassed many of these
processes, if present in rats.
Further considerations concern the signicance of
tracer uptake at the application site. Results that appear
inconsistent between studies may be due to confounding
effects produced by the injection device or the tracer itself.
Thus, in earlier studies in this eld, the relatively large
injection device (Tillet, 1992; Tillet et al., 1993; Wright
and Jennes, 1993; Simonian et al., 1999), the toxic effect of
Fluorogold (Dado et al., 1990; Tillet, 1992; Tillet et al.,
1993; Wright and Jennes, 1993), and/or the spaceoccupying nature of uorescent microspheres (Simonian
et al., 1999) might have caused damage to bers of passage, leading to ectopic uptake of the tracer. Although the
present study was designed to minimize such effects, they
cannot be excluded.
Telencephalon
BST and ventral lateral septum. The presence of
retrograde labelling in the medial division of the BST from
the site of GnRH perikarya and from the AVPV is consistent with its postulated role in processing pheromonal and
viscerosensory stimuli associated with reproduction (Canteras et al., 1995; Kippin et al., 2003). Anterograde tracing
studies have shown that projections from the medial division of the BST form a dense plexus of bers and terminals
in the AVPV and medial preoptic nucleus and that these
projections are particularly prominent in male rats (Hutton et al., 1998; Polston et al., 2004). In estrogen-primed,
ovariectomized rats, electrochemical stimulation of the
medial BST increases LH release; such stimulation in the
late morning on the day of proestrus advances the LH
surge (Beltramino and Taleisnik, 1980). In contrast,
equivalent stimulation of the lateral BST, where retrograde labelling was not observed in the present study, can
prevent the surge and ovulation (Beltramino and Taleisnik, 1980). Given these differentiated effects, it should
be noted that retrograde labelling in the BST from either
197
the GnRH perikarya site or the AVPV was most abundant

in the posteromedial division of the medial BST; the labelling from the AVPV predominated.
A high incidence of retrograde labelling from the AVPV,
comparable to that found in the posteromedial subdivision
of the medial BST, was found in another telencephalic
region, the ventral division of the lateral septum. A role
for the lateral septum in the modulation of motivated
behavior associated with defense or aggression has been
described (Clarke and Cummins, 1982; Kishore and Desiraju, 1990; Gavioli et al., 1999). Although a specic function for the ventral division of the lateral septum in estrous cycle control remains to be established, it should be
noted that moderate to high levels of ER and ER expression are found at this site and also in the medial
division of the BST (Shughrue et al., 1997; Li et al., 1997;
Yokosuka et al., 1997; Laamme et al., 1998; Zhang et al.,
2002; Hahn et al., 2003); this is consistent with a postulated role for these regions in estrogen modulation of
reproductive functions.
Amygdala. Moderate numbers of cells retrogradely
labelled from the AVPV were found in the medial amygdala; in this region, only sparse labelling was present from
the site of GnRH perikarya. In the amygdalohippocampal
area, the most caudal subdivision of the medial amygdala,
the incidence of labelling was high from the AVPV but
only moderate from the site of GnRH perikarya. ER and
ER are abundantly expressed in the posterodorsal subdivision of the medial amygdaloid nucleus and the anterolateral subdivision of the amygdalohippocampal area
(Shughrue et al., 1997; Li et al., 1997; Laamme et al.,
1998; Zhang et al., 2002). Anterograde tracing has shown
that each of these medial amygdaloid regions has extensive projections to the AVPV (Canteras et al., 1992a,
1995). Other sites targeted by projections from the amygdalohippocampal area include several that showed abundant retrograde labelling from the AVPV in the present
study, namely, the ventral lateral septal nucleus, medial
BST, ventral premammillary nucleus, and ventrolateral
ventromedial nucleus (Canteras et al., 1992a, 1995). Furthermore, a combined anterograde and retrograde tracing
study on male Syrian hamsters (Coolen and Wood, 1998)
has identied bidirectional connections between the posterodorsal subdivision of the medial amygdaloid nucleus
and the AVPV. In considering the signicance of medial
amygdala projections in reproduction-associated functions, it should be noted that electrolytic lesions of the
medial amygdala disrupt estrogen-dependent lordosis behavior and reduce mating-induced FOS expression in
GnRH neurons (Rajendren and Moss, 1993). Furthermore,
cytotoxic lesions at this site disinhibit maternal behavior
in rats (Sheehan et al., 2001). It is also noteworthy that
the medial amygdala is the principal telencephalic target
for pheromone-derived signals from the accessory olfactory system (Davis et al., 1978).
Subfornical organ. A low incidence of retrograde labelling was found in the subfornical organ from either the
GnRH perikarya site or the AVPV. Nuclear ER is
strongly expressed at this site (Yokosuka et al., 1997;
Shughrue et al., 1997; Laamme et al., 1998). Its ablation
disrupts the rat estrous cycle, producing a prolonged state
of diestrus (Limonta et al., 1981). The present ndings,
together with the previously reported evidence for an input to this circumventricular organ from the nucleus of
the solitary tract (Zardetto-Smith and Gray, 1987), raise
Fig. 2. Brain maps in the coronal plane showing the distribution

and relative abundance of retrograde labelling from a unilateral deposit of cholera toxin (CT) tracer (a; #22) at the site of gonadotrophinreleasing hormone (GnRH) perikarya or (b; #12) in the anteroventral
periventricular nucleus (AVPV). Quantication of retrograde labelling was performed by using a reticule; the mean number of labelled
cells in 200 200 m areas was determined (taking up to eight
measurements per neuroanatomical region). The results for each region were allocated into one of seven categories and represented
graphically. Five sizes of dot denote mean cell counts of 15, 6 10,
1120, 2135, or 36 60. A mean cell count greater than 60 is indicated by a star; blank regions reect an absence of labelling. These
data are mapped onto line diagrams modied from an atlas of the rat
brain (Paxinos and Watson, 1998). The approximate rostrocaudal
distance from Bregma is given for each level (corresponding to the
nearest gure in the atlas of Paxinos and Watson, 1998). The extent
of the CT tracer deposit is shown in diagrams (top left) traced from
photomicrographs. Scale bars 200 m in diagrams.
Figure 2
(Continued)
199
200
Figure 2
(Continued)
Figure 2
(Continued)
201
202
Fig. 3. Summary diagram illustrating the sites and relative

abundance (indicated by the thickness of the line and associated
circle) of cells retrogradely labelled by cholera toxin (CT) tracer
deposited either at the site of gonadotrophin-releasing hormone
(GnRH) perikarya, shown in black, or in the anteroventral periventricular nucleus (AVPV), shown in gray. The various brain regions
are displayed in a rostral to caudal direction from the tracer deposit. Sites of retrograde labelling are displayed separately for the
telencephalon, diencephalon, and combined mesencephalon and
rhombencephalon.
the possibility that it participates in relaying autonomic

and visceral information to inuence the reproductive cycle.
Diencephalon
Anteroventral periventricular nucleus. The observation of extensive retrograde labelling in the AVPV from
the vicinity of GnRH perikarya is consistent with previous
data that have demonstrated not only associations between the AVPV and GnRH perikarya but also a vital role
for the AVPV in the occurrence of the LH surge in rats
(Wiegand et al., 1978, 1980; Terasawa et al., 1980; Wiegand and Terasawa, 1982; Ronnekleiv and Kelly, 1988; Gu
and Simerly, 1997; Le et al., 1999, 2001; Simonian et al.,

1999; Petersen et al., 2003). Previous studies (Le at al.,
1999, 2001) indicate that the medial AVPV caudal to the
OVLT shows markedly elevated FOS expression at the
time of the LH surge. The labelling we observed from the
vicinity of GnRH perikarya encompassed the medial and
more lateral regions of the AVPV. This nucleus displays
abundant nuclear ER and ER (Kallo et al., 2001;
Shughrue and Merchenthaler, 2001); in the case of ER,
the expression is considerably more abundant at this site
in female rats than in male rats (Orikasa et al., 2002).
Recent ndings (Petersen et al., 2003) indicate that the
suppression by estrogen of GnRH gene expression may be
transiently overcome by estrogen-dependent signals relayed via the AVPV; this suggests a mechanism that may
be important for facilitating the LH surge. Furthermore,
there is evidence that the AVPV cells providing this relay
are multifunctional -aminobutyric acid (GABA)-ergic/
glutamatergic/neuropeptidergic neurons (Petersen et al.,
2003).
Medial preoptic nucleus. In the medial preoptic nucleus, retrograde labelling from the site of GnRH
perikarya or the AVPV was moderate to abundant; it was
present mostly in the medial subdivision of this nucleus,
where labelling from the AVPV predominated. In female
rats, the medial preoptic nucleus is a nodal point for the
control of lordosis (Pfaff et al., 1994). It is also a site with
abundant expression of ER and ER (Shughrue et al.,
1997; Li et al., 1997; Zhang et al., 2002). At the time of the
proestrous LH surge, the medial preoptic nucleus shows a
twofold increase in FOS expression (Herbison et al., 1995);
after mating, there is a fourfold increase in this signal
(Herbison et al., 1995). The current data are consistent
with ndings implicating this nucleus in the estrogensensitive relay of reproduction-related information to the
AVPV and to the site of GnRH perikarya in female rats
(Ronnekleiv and Kelly, 1986; Simonian et al., 1999; Li et
al., 2003).
Striohypothalamic nucleus. Retrograde labelling in
the striohypothalamic nucleus was abundant from the
AVPV but relatively sparse from the site of GnRH
perikarya. Earlier work has indicated a pathway to this
nucleus from the medial amygdala (Perez-Clausell et al.,
1989). Given that the striohypothalamic nucleus abuts the
dorsal boundary of the medial preoptic nucleus and contains ER (Mufson et al., 1999), the present ndings suggest an involvement in the control of reproductive functions that merits further investigation.
Median preoptic, anterodorsal preoptic, and parastrial nuclei. In a recent study (Thompson and Swanson,
2003), the median preoptic, anterodorsal preoptic, and
parastrial nuclei were identied as nodes in a highly interconnected six-node hypothalamic network in which the
other nodes were the AVPV, the anteroventral preoptic
nucleus (just lateral to the AVPV), and the DMH. This
network was proposed as a possible generator of the patterned activity necessary for, and characteristic of, autonomic and neuroendocrine control. In the present study,
ve nodes of this network, namely, the AVPV and the
median preoptic, anterodorsal preoptic, parastrial, and
dorsomedial nuclei, showed moderate to high levels of
retrograde labelling from the site of the GnRH perikarya
and/or the AVPV. In the median preoptic nucleus, labelling from the GnRH perikarya site was moderate but
sparse from the AVPV. In contrast, in the parastrial nu-

cleus, labelling from the AVPV was abundant but sparse
from the GnRH perikarya site. In the anterodorsal preoptic nucleus, a moderate level of labelling was seen from
both sites.
Given the input from the median preoptic nucleus to the
site of GnRH perikarya, it should be noted that some of
the neurons in this nucleus produce nitric oxide and may
be subject to inhibition by beta-endorphin via mu-opioid
receptors (Bouret et al., 2000). Nitric oxide has been implicated as a facilitator of GnRH release, particularly in
association with the LH surge (Bonavera et al., 1993b,
1994). Conversely, beta-endorphin may act as a brake on
GnRH release (Bonavera et al., 1993a), a brake that may
be released just before the onset of the LH surge (Allen et
al., 1988). Mu-opioid receptor-expressing nitric oxidesynthesizing cells are also found in the rostral preoptic
area, the site of GnRH perikarya (Bouret et al., 2000).
A strong projection to the AVPV from the parastrial
nucleus was detected. In this context, it is noteworthy that
the AVPV and the parastrial nucleus are both larger in
female rats than in male rats; in each case, the sexual
dimorphism is sensitive to perinatal alterations in circulating gonadal hormones (del Abril et al., 1990; Sumida et
al., 1993; Davis et al., 1996).
In the light of the moderate input from the anterodorsal
preoptic nucleus to the site of GnRH perikarya and to the
AVPV, it should be noted that a recent study (Peter et al.,
2004) demonstrated increased FOS expression in this nucleus following estrogen treatment during the day, but not
during the night. It remains to be established whether
this response is relevant to the timing of the proestrous
LH surge.
Suprachiasmatic nucleus, subparaventricular zone,
and lateroanterior hypothalamic nucleus. The SCN
has been implicated in both the timing and the production
of the LH surge. Thus, destruction of the SCN completely
blocks the daily LH surge induced by estrogen in ovariectomized rats (Coen and MacKinnon, 1980; Wiegand and
Terasawa, 1982); these lesions are effective without any
apparent damage to the preopticoinfundibular GnRH
pathway (Coen and MacKinnon, 1980). Such lesions also
disrupt the estrous cycle (Wiegand et al., 1978, 1980;
Wiegand and Terasawa, 1982). Nevertheless, acute damage to the SCN on the morning of proestrus is compatible
with subsequent ovulation (Terasawa et al., 1980). Furthermore, when estrogen priming is followed by a supraphysiological dose of progesterone, the evoked surge,
which is completely lost after ablation of the AVPV, is only
attenuated by SCN lesions (Wiegand et al., 1980; Wiegand
and Terasawa, 1982).
In the present study, a moderate incidence of retrograde
labelling was found predominantly within the dorsomedial division of the SCN from either the site of GnRH
perikarya or the AVPV. These injection sites produced a
low incidence of labelling in the ventromedial SCN and
only isolated cells in its dorsolateral region. In the ventromedial SCN, retrogradely labelled cells were slightly more
numerous from the AVPV than from the site of GnRH
perikarya; no such difference was observed in its dorsomedial or dorsolateral region. Anterograde tracing studies
have identied projections from the SCN to the AVPV and
to the rostral preoptic area in male (Watts et al., 1987) or
female (Watson et al., 1995) rats; furthermore, SCN efferents have been shown to make synaptic contact with
AVPV cells expressing nuclear ER (Watson et al., 1995).
203
In female hamsters, SCN efferents have been observed

making appositions with GnRH neurons (de la Iglesia et
al., 1995).
Our ndings corroborate those of a retrograde tracing
study in which most SCN projections to the medial preoptic area were found to arise from the dorsal region of the
SCN (Leak and Moore, 2001). A minor projection from the
ventrolateral SCN to the medial preoptic area was previously reported (Leak and Moore, 2001); consistent with
the latter observation, we found sparse retrograde labelling in the ventrolateral and central regions of the SCN,
but only in cases in which the site of the tracer deposit
included a part of the medial preoptic nucleus caudal to
the vicinity of the GnRH perikarya. In the subparaventricular zone, immediately above the dorsomedial SCN,
there was a higher level of retrograde labelling from the
site of GnRH perikarya than from the AVPV.
The dorsomedial subdivision of the SCN is characterized by numerous vasopressin-synthesizing neurons
(Moore et al., 2002). Efferents from these neurons have
been identied within the vicinity of the organum vasculosum of the lamina terminalis and in the AVPV (Hoorneman and Buijs, 1982; Leak and Moore, 2001; Watson et
al., 1995), but evidence for a direct vasopressinergic input
from the SCN to GnRH neurons is lacking. Various studies indicate a stimulatory role for vasopressin in relation
to the LH surge. Thus, when administered into the rostral
preoptic area after ablation of the SCN, vasopressin can
trigger a surge-like release of LH in estradiol-treated,
ovariectomized rats (Palm et al., 1999); in rats with an
intact SCN, the magnitude of LH release in response to
this treatment is subject to diurnal variation (Palm et al.,
2001). As a corollary, central administration of a vasopressin V1 receptor antagonist can attenuate the surge (Funabashi et al., 1999). In vitro studies on cocultures of the
preoptic area and SCN have shown that circadian release
of GnRH is dependent on the presence of estrogen and in
phase with the rhythm of AVP release (Funabashi et al.,
2000). We have recently reported (Kalamatianos et al.,
2004a) that mRNA for the V1a vasopressin receptor is
expressed in cells across the rostrocaudal extent of the
POA; some of these cells lie in close proximity to those
expressing GnRH mRNA, but coexpression of V1a and
GnRH mRNAs is detected only very rarely. The density of
V1a mRNA-expressing cells is particularly high within
the AVPV; at this site, its expression is elevated by estrogen treatment (Kalamatianos et al., 2004a). ERcontaining neurons in the AVPV are targets for efferents
from the dorsomedial SCN (Watson et al., 1995). Thus,
increased expression of V1a receptors in the AVPV by
estrogen may be a regulatory switch for indirect transduction of SCN signalling to GnRH neurons (Kalamatianos et
al., 2004a).
Although only at a very low incidence, cells retrogradely
labelled from the GnRH perikarya site were found in the
ventromedial SCN. Because this region of the SCN contains many vasoactive intestinal peptide (VIP)synthesizing cells (Kalamatianos et al., 2004b), the identied cells might contribute to the VIP-containing
projections that have been reported to form appositions on
GnRH neurons (van der Beek et al., 1993, 1997).
The lateroanterior hypothalamic nucleus, together with
the subparaventricular zone immediately dorsal to the
SCN, was one of the few diencephalic regions showing a
higher incidence of retrograde labelling from the site of
204
Figure 4
Figure 4
(Continued)
205
206
Fig. 4. Representative brighteld photomicrographs showing

perikarya immunoreactive for cholera toxin (CT) tracer in several
brain regions. Images a, c, e, g, i, k, m, o, and q show retrograde
labelling obtained from a CT deposit encompassing gonadotrophinreleasing hormone (GnRH) perikarya (as shown in Fig. 1a; #22);
images b, d, f, h, j, l, n, p, and r show retrograde labelling obtained
from a CT deposit in the AVPV (as shown in Fig. 1b; #12). The images
are paired to show a comparison of retrograde labelling obtained from
the GnRH or AVPV sites in corresponding brain regions. Outlines of
brain regions corresponding approximately to those delineated in an
atlas of the rat brain (Paxinos and Watson, 1998) are represented as
dashed lines. a,b: Retrograde labelling in the ventral part of the
lateral septal nucleus (LSV). c,d: Retrograde labelling in posterior
subdivisions of the medial bed nucleus of the stria terminalis. e,f:
Retrograde labelling in the suprachiasmatic nucleus (SCN) and lateroanterior hypothalamic nucleus (LA). g,h: Retrograde labelling in
the mid rostrocaudal region of the arcuate nucleus. i,j: Retrograde
labelling in the posterodorsal subdivision of the medial amygdaloid
nucleus (MePD). k,l: Retrograde labelling in the posterior subdivision
of the ventrolateral division of the ventromedial hypothalamic nucleus (VMHVLp). m,n: Retrograde labelling in the caudal arcuate
nucleus and ventral premammillary nucleus (PMV). o,p: Retrograde
labelling in the anterolateral subdivision of the amygdalohippocampal area (AHiAL). q,r: Retrograde labelling in the lateral parabrachial nucleus, prominent from the GnRH site at this level in the
central division (LPBC). The sections shown in b, d, f, h, j, l, n, p, and
r were treated with a Nissl counterstain (which has been partially
suppressed prior to conversion to gray scale). Scale bars 100 m.

GnRH perikarya than from the AVPV. A role for the
lateroanterior hypothalamic nucleus in the control of the
ovulatory cycle has not been established; nevertheless,
suckling-induced lactation in rats is associated with an
increase in prolactin receptor expression at that site (Pi
and Voogt, 2000). Because exogenous prolactin can suppress plasma LH (Carter et al., 1983), the present ndings
suggest an indirect route by which this hormone might
inuence GnRH neurons, if it can cross the blood brain
barrier during lactation.
Retrochiasmatic area. Retrograde labelling within
the retrochiasmatic area was only sparse from the AVPV
but was moderate from the site of GnRH perikarya. Electrolytic lesioning of this area in rats blocks the inhibition
of LH release following electrical stimulation of the dorsal
raphe nucleus (Arendash and Gallo, 1979). In hamsters,
retrochiasmatic efferents make appositions with GnRH
perikarya and with ER-immunopositive neurons in the
medial BST and in the septohypothalamic nucleus (de la
Iglesia et al., 1995) [which is synonymous with the striohypothalamic nucleus in the present study (according to
Paxinos and Watson, 1998]; as shown here, the latter two
sites have extensive projections to the AVPV. Thus, although the input to the AVPV from the retrochiasmatic
area appears sparse, retrochiasmatic efferents may inuence the AVPV via projections from the medial BST and
striohypothalamic nucleus to that site; these indirect
pathways may involve estrogen-responsive neurons.
Paraventricular hypothalamic nucleus. Only a low
incidence of retrograde labelling was found within the
PVN from either the site of GnRH perikarya or the AVPV.
The PVN has received considerable attention regarding
its possible involvement in the control of LH release; much
of this has focused on the CRH-synthesizing neurons in
the medial parvocellular subdivision of the PVN. Various
stressors suppress the pulsatile release of LH in rats
(Blake, 1975; Rivier et al., 1986; Dong et al., 1994; Nagatani et al., 1996; Goubillon and Thalabard, 1996; Cagampang et al., 1997, 1999; Tsukahara et al., 1999); such
suppression may be mediated by central actions of CRH
(Rivier and Vale, 1984; Ono et al., 1984; Rivier et al., 1986;
Petraglia et al., 1987; Maeda et al., 1994; Roozendaal et
al., 1996; Tsukahara et al., 1999). Our present ndings of
sparse retrograde labelling within the medial parvocellular PVN are consistent with the notion that CRH neurons
at this site do not inuence the reproductive axis by direct
input to GnRH perikarya; this issue has been addressed in
a previous report in which we presented evidence against
the existence of direct CRH projections from the PVN to
the vicinity of GnRH cells bodies in female rats (Hahn et
al., 2003). In this context, it should be noted that several
studies have indicated that CRH may not be required for
stress-induced LH suppression (Rivest and Rivier, 1991;
Akema et al., 1996; Jeong et al., 1999).
Arcuate nucleus. A predominantly moderate level of
retrograde labelling was found across the rostrocaudal
extent of the arcuate nucleus from the site of GnRH
perikarya or the AVPV. Numerous previous studies have
indicated a role for the arcuate nucleus in the control of
LH release and fertility. Electrophysiological recordings
from this region in rats show a close temporal association
between multiunit activity volleys and LH pulses (Goubillon et al., 1995, 1999). Furthermore, deleterious effects on
reproductive physiology have been observed following cytotoxic lesioning of this nucleus by neonatal monosodium
207
glutamate; these effects include reduced ovarian weight

(Badger et al., 1982) and a delay in the onset of puberty
(MacDonald and Wilkinson, 1990) in rats, a loss of estrous
cycles in hamsters (Lamperti and Blaha, 1980), and a
reduced rate of conception in mice (Olney, 1969). For rats,
such lesions have been reported to lead to a slight reduction in mean plasma LH (Greeley et al., 1978; Sridaran et
al., 1981) and to perturb, but not abolish, pulsatile LH
release (Sridaran et al., 1981). The rat arcuate nucleus
displays abundant expression of ER but only weak expression of ER (Shughrue et al., 1997; Li et al., 1997;
Laamme et al., 1998; Zhang et al., 2002). The present
ndings are consistent with those of previous retrograde
tracing studies demonstrating projections from the arcuate nucleus to the region of GnRH perikarya (Simonian et
al., 1999) or to the medial preoptic area (Horvath et al.,
1992), and expand on those reports by identifying projections from the entire rostrocaudal extent of this nucleus to
the site of GnRH perikarya and the AVPV.
Dorsomedial and ventromedial nuclei. Retrograde
labelling of the DMH from the site of GnRH perikarya or
the AVPV was observed; this was particularly abundant
in its ventral division, where labelling from the GnRH
perikarya site predominated. The DMH participates in
drinking, feeding, and body weight regulation (Bellinger
and Bernardis, 2002). A specic role for the DMH in the
central control of reproduction-related functions remains
to be established. Nevertheless, electrical stimulation of
the DMH has been shown to reduce plasma LH levels in
rats; this is associated with a prolongation of the interval
between LH pulses (Gallo, 1981). Given this inhibitory
response, it may be signicant that the DMH is strongly
activated by various stressors (Cullinan et al., 1995, 1996;
Canteras et al., 1997; Li and Sawchenko, 1998). Thus, the
inputs from the DMH to the site of GnRH perikarya or the
AVPV may be involved in modulating LH release under
conditions of stress.
Within the posterior pole of the ventrolateral VMH, a
particularly high incidence of retrogradely labelled cells
was found from the AVPV; from the site of the GnRH
perikarya, the incidence was lower. Elsewhere in the
VMH, retrograde labelling from either of these sites was
sparse. In contrast to the DMH, a clear reproductionrelated role has been identied for the VMH. Thus, lordosis can be elicited by stimulation of the VMH (Pfaff and
Sakuma, 1979b) and is associated with FOS expression in
its ventrolateral subdivision (Wedemeyer et al., 1999);
lesions of the VMH, particularly its ventrolateral subdivision, disrupt this copulatory reex (Pfaff and Sakuma,
1979a). Induction of lordosis is regulated by the stage of
the estrous cycle and may be facilitated by central actions
of GnRH (Moss and McCann, 1973; Pfaff, 1973; Mani et
al., 1994); at least one site for such actions has been
proposed: the mesencephalic periaqueductal gray (PAG).
Thus, the incidence of lordotic behavior is enhanced by
GnRH infusion into the PAG (Riskind and Moss, 1983), a
site containing GnRH-immunoreactive axons (Merchenthaler et al., 1984). Tract tracing studies have identied
a projection from the VMH to the PAG (Saper et al., 1976;
Beitz, 1982) and divergent collaterals from the ventrolateral VMH that project to both the medial preoptic nucleus
and the PAG (Akesson et al., 1994). Abundant expression
of ER (Shughrue et al., 1997; Laamme et al., 1998), but
only weak expression of ER (Laamme et al., 1998), is
found in the ventrolateral VMH.
208
Given that the VMH, particularly its posterior subdivision, is crucial for copulatory behavior in female rats, the
inputs to the AVPV and to the region of the GnRH
perikarya from the posterior ventrolateral VMH may be
involved in integrating neuroendocrine and behavioral
systems. Our ndings are consistent with the results of a
previous retrograde tracing study in female rats in which
injections of Fluorogold centered in the periventricular
zone of the rostral preoptic area, but with sufcient spread
to include the adjacent site of the GnRH perikarya and
AVPV, resulted in substantial retrograde labelling of the
posterior ventrolateral VMH (Hoffman et al., 1996). In the
study by Hoffman et al. (1996), the posterior pole of the
VMH was identied by the presence of enkephalinergic
immunoreactive perikarya in this region, some of which
were found to colocalize the Fluorogold tracer. Furthermore, a recent comparative study in male and female rats
found the posterior pole of the VMHVL to be twice as
large, and to contain 40% more ER positive neurons, in
the females (Schutterbrandt et al., 2004).
Tract tracing studies in sheep have also shown projections from the VMH to rostral periventricular regions and
close appositions between anterogradely lled processes
and GnRH perikarya (Goubillon et al., 2002). In apparent
contrast to these ndings, an anterograde tracing study
observed only sparse projections from the ventrolateral
division of the VMH to the AVPV in rats (Canteras et al.,
1994); nevertheless, those ndings are not inconsistent
with the present results, because the origin of the projections investigated was slightly dorsal and rostral to the
site of abundant retrograde labelling identied here.
Caudal diencephalon. Caudal to the VMH and DMH,
the remaining diencephalic region showing abundant retrograde labelling was the ventral premammillary nucleus
(PMV). The high incidence of retrograde labelling from the
AVPV in this region is in striking contrast to the low
incidence from the site of GnRH perikarya. Our ndings
are consistent with those of an anterograde tracing study
that identied a substantial projection from the PMV to
rostral periventricular regions of the hypothalamus, including the AVPV (Canteras et al., 1992b). The PMV also
provides dense innervation of the posteromedial subdivision of the amygdalohippocampal area, posterodorsal subdivision of the medial amygdaloid nucleus, ventrolateral
division of the VMH, medial division of the medial preoptic nucleus, medial BST, and ventral division of the lateral
septal nucleus (Canteras et al., 1992b); each of these sites
is shown, in the current study, to provide substantial
projections to the AVPV and to a lesser extent to the site
of GnRH perikarya. Prominent among the regional
sources of inputs to the PMV are the medial amygdala, the
medial BST, and the sexually dimorphic components of
the accessory olfactory system (Numan and Numan, 1996;
Guillamon and Segovia, 1997). Information arriving at the
PMV via the accessory olfactory system appears to have
particular signicance in relation to the control of LH
release; thus, bilateral PMV lesions prevent male
pheromone-induced stimulation of LH release in ovariectomized, estrogen-primed rats (Beltramino and Taleisnik,
1985). Furthermore, a unilateral PMV lesion prevents LH
release in response to ipsilateral, but not contralateral,
stimulation of the BST or medial amygdala (Beltramino
and Taleisnik, 1985). ER is expressed at a moderate
incidence in the PMV (Shughrue et al., 1997). It should
also be noted that the abundant melatonin binding sites in

this nucleus have been implicated in the stimulation of LH
release in sheep (Malpaux et al., 1998).
Cells retrogradely labelled from the GnRH perikarya
site or the AVPV were found at a moderate to low incidence extending from the lower border of the ventral premammillary nucleus, through the lateral and medial posterior
arcuate
nucleus
and
into
the
dorsal
tuberomammillary nucleus; these regions encompass, the
E3 and E4 subgroups of histaminergic neurons (Inagaki et
al., 1990). There was also a sparse incidence of labelling
from the GnRH perikarya site in the region of the E5
neurons (the tuber cinereum area) and E2 neurons (the
marginal zone near the ventral tuberomammillary nucleus). The histaminergic cells in these populations show a
very considerable range in cell size (Ericson et al., 1987);
in the present study, the local magnocellular neurons
were not seen to be labelled. The possibility that cells
found in the E2E5 regions might have included those
that synthesize histamine is suggested by the discovery of
histamine-immunoreactive axons in apposition to GnRH
neurons (Fekete et al., 1999). Furthermore, we have
shown that the LH surge can be blocked by central administration of an antagonist to H1 receptors, but not to
H2 receptors (Fekete et al., 1999).
Within the medial supramammillary nucleus, a moderate incidence of retrograde labelling was found from the
AVPV and a slightly lower incidence from the site of
GnRH perikarya. The supramammillary nucleus is implicated in maternal physiology; thus, an increase in FOS
expression occurs at this site in parturient rats (Lin et al.,
1998), and its electrolytic ablation abolishes oxytocininduced facilitation of the milk-ejection reex (Wakerley
and Cosgrave, 2002). In this context, it may be speculated
that projections from the supramammillary nucleus to the
site of GnRH perikarya, and more prominently to the
AVPV, participate in the suppression of fertility associated with parturient and postpartum physiology. At this
site, ER is expressed at a moderate incidence and ER at
a low incidence (Shughrue et al., 1997).
Mesencephalon and rhombencephalon

Periaqueductal gray, locus coeruleus, and median
and dorsal raphe nuclei. A low incidence of retrograde
labelling was found in the ventrolateral PAG from the site
of GnRH perikarya and from the AVPV. Because the PAG
has an important role in lordosis (Sakuma and Pfaff, 1979;
Hennessey et al., 1990), a role that seems to involve local
actions of GnRH (Riskind and Moss, 1983), the projections
from the PAG to the site of GnRH perikarya might inuence the contributions made by this peptide to copulatory
behavior. At this site, expression of ER is moderate and
that of ER is low (Shughrue et al., 1997). It is noteworthy
that the PAG receives an input from the locus coeruleus
(Luppi et al., 1995) and that the border of the locus coeruleus, which contains dendrites of noradrenergic neurons,
receives projections from the ventrolateral PAG (Mihaly et
al., 2001). In the present study, sparse retrograde labelling was observed in the locus coeruleus, but only when
the tracer deposit included the medial preoptic nucleus
caudal to the site of GnRH perikarya; this nding appears
to be consistent with the reported decrease in noradrenergic input to the medial preoptic area following electrolytic lesioning of the locus coeruleus (Anselmo-Franci et
al., 1997) and with the results of a previous retrograde
tracing study (Wright and Jennes, 1993). Despite the ab-

sence of retrograde labelling in the locus coeruleus from
the AVPV or the site of GnRH perikarya, it should be
noted that lesioning this site has been reported to block
the LH surge (Anselmo-Franci et al., 1997; Helena et al.,
2002; Martins-Afferri et al., 2003) and ovulation (Franci
and Antunes-Rodrigues, 1985).
A low incidence of retrograde labelling was found in the
median and dorsal raphe nuclei from the AVPV and the site
of GnRH perikarya. Distinct reproductive roles for these
nuclei are suggested by functional studies. Thus, electrochemical stimulation of the median raphe nucleus in rats on
the afternoon of proestrus blocks ovulation (Barofsky, 1979;
Morello and Taleisnik, 1985). This effect can also be produced by electrolytic lesioning of the dorsal, but not the
median, raphe nucleus (Morello and Taleisnik, 1985); there
is evidence that this blocking of ovulation may involve removal of a 5-hydroxytryptamine (5-HT) projection from the
dorsal raphe nucleus to the locus coeruleus (Morello and
Taleisnik, 1988). Electrical stimulation of the dorsal raphe
nucleus inhibits LH release in ovariectomized rats (Arendash and Gallo, 1978), but not in the presence of estrogen
(Kitts and Johnson, 1986). There is a sparse expression of
ER and ER in the raphe nuclei (Shughrue et al., 1997).
The possibility that inputs to the GnRH perikarya from
raphe nuclei release 5-HT is supported by the discovery of
contacts between GnRH perikarya and axonal boutons containing tritiated 5-HT (Kiss and Halasz, 1985). Furthermore, 5-HT-immunoreactive neurons expressing the mRNA
for ER have been shown to project from the dorsal raphe
nucleus to the medial preoptic area (Lu et al., 2001). In this
context, it should be noted that several previous studies have
suggested a critical involvement of 5-HT in the control of LH
release (Coen and MacKinnon, 1979, 1980; Coen et al.,
1980).
Lateral parabrachial nucleus. The abundance of
cells retrogradely labelled from the GnRH perikarya site
in the central and, to a lesser extent, the dorsal and
superior lateral parabrachial nucleus is in contrast to
their incidence in other brainstem regions, where relatively few labelled cells were found. The parabrachial nucleus is a complex of nuclei that integrate and relay visceral and autonomic input from the caudal brainstem and
spinal cord to higher brain areas. Located in the dorsal
pons, it is divided by the superior cerebellar peduncle into
two parts: the lateral part, which has been implicated in
autonomic and behavioral responses to pain or other aversive stimuli (Mizusawa et al., 1995; Gauriau and Bernard,
2002; Jurgens, 2002), and the medial part, which has been
implicated in gustatory and appetitive functions (Spector,
1995; Reilly, 1999; Bray, 2000).
A role for the parabrachial nucleus in the control of the
ovulatory cycle has not been established. Nevertheless,
available evidence suggests that reproductive status in
female rats is associated with changes in the activity of
this nucleus. Thus, its electrical activity in response to a
bitter taste is increased following ovariectomy (Di Lorenzo
and Monroe, 1990), and local FOS expression in response
to hemorrhage is greater in nulliparous than in pregnant
rats (Jaworski et al., 2002). Given that a major projection
to the central subdivision of the lateral parabrachial nucleus arises from the nucleus of the solitary tract (Herbert
et al., 1990), the terminus of vagal afferents, it may be
pertinent to note that severance of the gastric vagus restores LH pulses that have been suppressed by 48 hours of
209
fasting in ovariectomized, estrogen-replaced rats (Cagampang et al., 1992).

The present ndings are consistent with those of anterograde tracing studies that demonstrated substantial projections from the lateral parabrachial nucleus (in particular its central, dorsal, and external subdivision) to the
rostral medial preoptic area and adjacent periventricular
region (Krukoff et al., 1993; Alden et al., 1994; Bester et
al., 1997). Expression of ER (Shughrue et al., 1997; Hahn
et al., 2003) and ER (Shughrue et al., 1997) occurs at a
relatively low incidence in the lateral parabrachial nucleus of rats. In sheep, retrograde tracing from the region
of GnRH perikarya has revealed numerous cells in the
lateral parabrachial nucleus; very few corresponded to the
local neurons containing ER-immunoreactive nuclei
(Scott et al., 1999).
In addition to receiving major projections from the nucleus of the solitary tract and area postrema, the lateral
parabrachial nucleus is also targeted by the locus coeruleus (Luppi et al., 1995). Anterograde tracing studies
have described projections from the dorsal raphe nucleus
to both the lateral parabrachial nucleus and the locus
coeruleus (Vertes and Kocsis, 1994); the former projection
involves a substantial non-5-HT component and collaterals to the parvicellular PVN (Petrov et al., 1992). Projections from the ventrolateral PAG to the central and dorsal
lateral parabrachial nucleus have also been identied
(Krout et al., 1998). Major descending projections from the
lateral hypothalamus, arcuate nucleus, retrochiasmatic
area, BST, and central nucleus of the amygdala target the
lateral parabrachial nucleus (Moga and Gray, 1985; Moga
et al., 1989, 1990). Thus, extensive pathways connecting
the lateral parabrachial nucleus with the hypothalamus
have been identied, but little is currently known about
their possible roles in relaying information specically
relevant to reproduction.
Ventrolateral medulla and solitary tract nucleus.
Previous studies identied projections from the ventrolateral medulla and nucleus of the solitary tract to the vicinity of the GnRH perikarya in the rat (CastaneyraPerdomo et al., 1992; Wright and Jennes, 1993; Simonian
et al., 1999); projections have also been reported to originate in the C3 adrenergic cell group (Castaneyra-Perdomo
et al., 1992), a nding that was not conrmed with the
very small injection volumes in the present study. Analysis of the neurochemical phenotype of cells back-lled
from the vicinity of the GnRH perikarya (Wright and
Jennes, 1993; Simonian et al., 1999) identied noradrenergic projections from the nucleus of the solitary tract (A2)
and the ventrolateral medulla (A1). Nuclear ER was
found in some of the A2 noradrenaline cells retrogradely
labelled from the rostral preoptic area; this was not the
case for the ventrolateral medulla A1 cells (Simonian et
al., 1999). It should be noted that the incidence of expression for both ER and ER is higher in the region of the
nucleus of the solitary tract than in the ventrolateral
medulla (Shughrue et al., 1997). It remains to be established whether the relatively few cells at these sites that
were back-lled from the region of GnRH perikarya in the
present study correspond to local noradrenaline and/or
adrenaline neurons; such information would complement
the ndings of earlier studies that assessed the roles of
these catecholamines in the regulation of LH release
(Coen et al., 1983, 1985; Coen and Coombs, 1983; Coombs
and Coen, 1983; Coen and Gallo, 1986).
210
CONCLUSIONS
This study has investigated the sources of projections to
parts of the preoptic area that are indispensable for the
maintenance of the rat ovulatory cycle: the site containing
GnRH perikarya and the AVPV. Reviewing these data in
the context of previous functional or neuroanatomical
ndings indicates that many of the identied regions are
implicated in reproduction-related processes. The cataloguing of the similarities and differences in the inputs to
the site of GnRH perikarya and to the AVPV, as presented
here, may help to elucidate the mechanisms involved in
generating or suppressing the reproductive cycle and in
integrating its components with associated sensory, behavioral, and homeostatic processes.
LITERATURE CITED
Akema T, Chiba A, Shinozaki R, Oshida M, Kimura F, Toyoda J. 1996.
Acute immobilization stress and intraventricular injection of CRF suppress naloxone-induced LH release in ovariectomized estrogen-primed
rats. J Neuroendocrinol 8:647 652.
Akesson TR, Ulibarri C, Truitt S. 1994. Divergent axon collaterals originate in the estrogen receptive ventromedial nucleus of hypothalamus
in the rat. J Neurobiol 25:406 414.
Alden M, Besson JM, Bernard JF. 1994. Organization of the efferent
projections from the pontine parabrachial area to the bed nucleus of the
stria terminalis and neighboring regions: a PHA-L study in the rat.
J Comp Neurol 341:289 314.
Allen LG, Hahn E, Caton D, Kalra SP. 1988. Evidence that a decrease in
opioid tone on prestrus changes the episodic pattern of luteinizing
hormone (LH) secretion: implications in the preovulatory LH hypersecretion. Endocrinology 122:1004 1013.
Anselmo-Franci JA, Franci CR, Krulich L, Antunes-Rodrigues J, McCann
SM. 1997. Locus coeruleus lesions decrease norepinephrine input into
the medial preoptic area and medial basal hypothalamus and block the
LH, FSH and prolactin preovulatory surge. Brain Res 767:289 296.
Arendash GW, Gallo RV. 1978. Serotonin involvement in the inhibition of
episodic luteinizing hormone release during electrical stimulation of
the midbrain dorsal raphe nucleus in ovariectomized rats. Endocrinology 102:1199 1206.
Arendash GW, Gallo RV. 1979. Effect of lesions in the suprachiasmatic
nucleus-retrochiasmatic area on the inhibition of pulsatile LH release
induced by electrical stimulation of the midbrain dorsal raphe nucleus.
Neuroendocrinology 28:349 357.
Badger TM, Millard WJ, Martin JB, Rosenblum PM, Levenson SE. 1982.
Hypothalamic-pituitary function in adult rats treated neonatally with
monosodium glutamate. Endocrinology 111:20312038.
Barofsky AL. 1979. Median raphe stimulation and sham procedures inhibit
the LH surge. Neuroendocrinology 28:358 370.
Bellinger LL, Bernardis LL. 2002. The dorsomedial hypothalamic nucleus
and its role in ingestive behavior and body weight regulation. Lessons
learned from lesioning studies. Physiol Behav 76:431 442.
Beltramino C, Taleisnik S. 1980. Dual action of electrochemical stimulation of the bed nucleus of the stria terminalis on the release of LH.
Beltramino C, Taleisnik S. 1985. Ventral premammillary nuclei mediate
pheromonal-induced LH release stimuli in the rat. Neuroendocrinology
41:119 124.
Berk ML, Finkelstein JA. 1981. Afferent projections to the preoptic area
and hypothalamic regions in the rat brain. Neuroscience 6:16011624.
Bester H, Besson JM, Bernard JF. 1997. Organization of efferent projections from the parabrachial area to the hypothalamus: a Phaseolus
vulgaris-leucoagglutinin study in the rat. J Comp Neurol 383:245281.
Beitz AJ. 1982. The organization of afferent projections to the midbrain
periaqueductal gray of the rat. Neuroscience 7:133159.
Blake CA. 1975. Effects of stress on pulsatile luteinizing hormone release
in ovariectomized rats. Proc Soc Exp Biol Med 148:813 815.
Bleier R, Byne W, Siggelkow I. 1982. Cytoarchitectonic sexual dimorphisms of the medial preoptic and anterior hypothalamic areas in
guinea pig, rat, hamster, and mouse. J Comp Neurol 212:118 130.
Bonavera JJ, Kalra SP, Kalra PS. 1993a. Evidence that luteinizing hor-
mone suppression in response to inhibitory neuropeptides, betaendorphin, interleukin-1 beta, and neuropeptide-K, may involve excitatory amino acids. Endocrinology 133:178 182.
Bonavera JJ, Sahu A, Kalra PS, Kalra SP. 1993b. Evidence that nitric
oxide may mediate the ovarian steroid-induced luteinizing hormone
surge: involvement of excitatory amino acids. Endocrinology 133:2481
2487.
Bonavera JJ, Sahu A, Kalra PS, Kalra SP. 1994. Evidence in support of
nitric oxide (NO) involvement in the cyclic release of prolactin and LH
surges. Brain Res 660:175179.
Bouret S, Prevot V, Croix D, Viltart O, Stefano GB, Mitchell V, Beauvillain
JC. 2000. Mu opioid receptor mRNA expression in neuronal nitric oxide
synthase-immunopositive preoptic area neurons. Brain Res Mol Brain
Res 80:46 52.
Bray GA. 2000. Afferent signals regulating food intake. Proc Nutr Soc
59:373384.
Brody MJ, Johnson AK. 1980. Role of the anteroventral third ventricle
region in uid and electrolyte balance, arterial pressure regulation and
hypertension. In: Martini L Ganong WF, editors. Frontiers in neuroendocrinology. New York: Raven. p 249 292.
Cagampang FR, Maeda K, Ota K. 1992. Involvement of the gastric vagal
nerve in the suppression of pulsatile luteinizing hormone release during acute fasting in rats. Endocrinology 130:30033006.
Cagampang FR, Cates PS, Sandhu S, Strutton PH, McGarvey C, Coen CW,
OByrne KT. 1997. Hypoglycaemia-induced inhibition of pulsatile luteinizing hormone secretion in female rats: role of estradiol, endogenous opioids and the adrenal medulla. J Neuroendocrinol 9:867 872.
Cagampang FR, Strutton PH, Goubillon ML, Coen CW. 1999. Adrenomedullectomy prevents the suppression of pulsatile luteinising
hormone release during fasting in female rats. J Neuroendocrinol 11:
429 433.
Campbell RE, Han SK, Herbison AE. 2005. Biocytin lling of adult
gonadotropin-releasing hormone neurons in situ reveals extensive,
spiny, dendritic processes. Endocrinology 146:11631169.
Canteras NS, Simerly RB, Swanson LW. 1992a. Connections of the posterior nucleus of the amygdala. J Comp Neurol 324:143179.
Canteras NS, Simerly RB, Swanson LW. 1992b. Projections of the ventral
premammillary nucleus. J Comp Neurol 324:195212.
Canteras NS, Simerly RB, Swanson LW. 1994. Organization of projections
from the ventromedial nucleus of the hypothalamus: a Phaseolus
vulgaris-leucoagglutinin study in the rat. J Comp Neurol 348:4179.
Canteras NS, Simerly RB, Swanson LW. 1995. Organization of projections
from the medial nucleus of the amygdala: a PHAL study in the rat.
J Comp Neurol 360:213245.
Canteras NS, Chiavegatto S, Valle LE, Swanson LW. 1997. Severe reduction of rat defensive behavior to a predator by discrete hypothalamic
chemical lesions. Brain Res Bull 44:297305.
Carter DA, Lakhani S, Whitehead SA. 1983. Characterization of the inhibitory effects of hyperprolactinaemia on the mechanism controlling
LH secretion in chronically ovariectomized rats. J Reprod Fertil 69:57
64.
Castaneyra-Perdomo A, Perez-Delgado MM, Montagnese C, Coen CW.
1992. Brainstem projections to the medial preoptic region containing
the luteinizing hormone-releasing hormone perikarya in the rat. An
immunohistochemical and retrograde transport study. Neurosci Lett
139:135139.
Chiba T, Murata Y. 1985. Afferent and efferent connections of the medial
preoptic area in the rat: a WGA-HRP study. Brain Res Bull 14:261
272.
Clarke IJ, Cummins JT. 1982. The temporal relationship between gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) secretion in ovariectomized ewes. Endocrinology 111:17371739.
Coen CW, Coombs MC. 1983. Effects of manipulating catecholamines on
the incidence of the preovulatory surge of luteinizing hormone and
ovulation in the rat: evidence for a necessary involvement of hypothalamic adrenaline in the normal or midnight surge. Neuroscience
10:187206.
Coen CW, Gallo RV. 1986. Effects of various inhibitors of phenylethanolamine N-methyltransferase on pulsatile release of luteinizing hormone in ovariectomized rats. J Endocrinol 111:5159.
Coen CW, MacKinnon PCB. 1979. Serotonin involvement in the control of
phasic luteinizing hormone release in the rat: evidence for a critical
period. J Endocrinol 82:105113.
Coen CW, MacKinnon PC. 1980. Lesions of the suprachiasmatic nuclei and
the serotonin-dependent phasic release of luteinizing hormone in the

rat: effects on drinking rhythmicity and on the consequences of preoptic
area stimulation. J Endocrinol 84:231236.
Coen CW, Franklin M, Laynes RW, MacKinnon PCB. 1980. Effects of
manipulating serotonin on the incidence of ovulation in the rat. J
Endocrinol 87:195201.
Coen CW, Coombs MC, Wilson PMJ, Clement EM, MacKinnon PCB. 1983.
Possible resolution of a paradox concerning the use of
p-chlorophenylalanine and 5-hydroxytryptophan: evidence for a mode
of action involving adrenaline in manipulating the surge of luteinizing
hormone in rats. Neuroscience 8:583591.
Coen CW, Simonyi A, Fekete MIK. 1985. Effect of phenylethanolamine
N-methyltransferase inhibition on serum concentrations of luteinizing
hormone, follicle stimulating hormone and prolactin in pro-estrous and
ovariectomized rats. Brain Res 343:383387.
Coen CW, Montagnese C, Opacka-Juffry J. 1990. Coexistence of gonadotrophin releasing hormone and galanin: immunohistochemical and
functional studies. J Neuroendocrinol 2:107111.
Contreras CM, Molina M, Saavedra M, Martinez-Mota L. 2000. Lateral
septal neuronal ring rate increases during prestrus-estrus in the rat.
Physiol Behav 68:279 284.
Coolen LM, Wood RI. 1998. Bidirectional connections of the medial amygdaloid nucleus in the Syrian hamster brain: simultaneous anterograde
and retrograde tract tracing. J Comp Neurol 399:189 209.
Coombs MC, Coen CW. 1983. Adrenaline turnover rates in the medial
preoptic area and mediobasal hypothalamus in relation to the release
of luteinizing hormone in female rats. Neuroscience 10:207210.
Cullinan WE, Herman JP, Battaglia DF, Akil H, Watson SJ. 1995. Pattern
and time course of immediate early gene expression in rat brain following acute stress. Neuroscience 62:477505.
Cullinan WE, Helmreich DL, Watson SJ. 1996. Fos expression in forebrain
afferents to the hypothalamic paraventricular nucleus following swim
stress. J Comp Neurol 368:88 99.
Dado RJ, Burstein R, Cliffer KD, Giesler GJ. 1990. Evidence that FluoroGold can be transported avidly through bers of passage. Brain Res
533:329 333.
Davis BJ, Macrides F, Youngs WM, Schneider SP, Rosene DL. 1978.
Efferents and centrifugal afferents of the main and accessory olfactory
bulbs in the hamster. Brain Res Bull 3:59 72.
Davis EC, Shryne JE, Gorski, RA. 1996. Structural sexual dimorphisms in
the anteroventral periventricular nucleus of the rat hypothalamus are
sensitive to gonadal steroids perinatally, but develop peripubertally.
Neuroendocrinology 63:142148.
Day TA, Blessing W, Willoughby JO. 1980. Noradrenergic and dopaminergic projections to the medial preoptic area of the rat. A combined
horseradish peroxidase/catecholamine uorescence study. Brain Res
193:543548.
de la Iglesia HO, Blaustein JD, Bittman EL. 1995. The suprachiasmatic
area in the female hamster projects to neurons containing estrogen
receptors and GnRH. Neuroreport 6:17151722.
del Abril A, Segovia S, Guillamon A. 1990. Sexual dimorphism in the
parastrial nucleus of the rat preoptic area. Brain Res Dev Brain Res
52:1115.
Di Lorenzo PM, Monroe S. 1990. Taste responses in the parabrachial pons
of ovariectomized rats. Brain Res Bull 25:741748.
Dong Q, Bergendahl M, Huhtaniemi I, Handelsman DJ. 1994. Effect of
undernutrition on pulsatile luteinizing hormone (LH) secretion in castrate and intact male rats using an ultrasensitive immunouorometric
LH assay. Endocrinology 135:745750.
Ericson H, Watanabe T, Kohler C. 1987. Morphological analysis of the
tuberomammillary nucleus in the rat brain: delineation of subgroups
with antibody against L-histidine decarboxylase as a marker. J Comp
Neurol 263:124.
Fekete CS, Strutton PH, Cagampang FR, Hrabovszky E, Kallo I, Shughrue
PJ, Dobo E, Mihaly E, Baranyi L, Okada H, Panula P, Merchenthaler
I, Coen CW, Liposits ZS. 1999. Estrogen receptor immunoreactivity is
present in the majority of central histaminergic neurons: evidence for
a new neuroendocrine pathway associated with luteinizing hormonereleasing hormone-synthesizing neurons in rats and humans. Endocrinology 140:4335 4341.
Franci JA, Antunes-Rodrigues J. 1985. Effect of locus ceruleus lesion on
luteinizing hormone secretion under different experimental conditions.
Funabashi T, Aiba S, Sano A, Shinohara K, Kimura F. 1999. Intracerebroventricular injection of arginine-vasopressin V1 receptor antagonist
211
attenuates the surge of luteinizing hormone and prolactin secretion in

prestrous rats. Neurosci Lett 260:37 40.
Funabashi T, Shinohara K, Mitsushima D, Kimura F. 2000. Gonadotropinreleasing hormone exhibits circadian rhythm in phase with argininevasopressin in co-cultures of the female rat preoptic area and suprachiasmatic nucleus. J Neuroendocrinol 12:521528.
Gallo RV. 1981. Effect of electrical stimulation of the dorsomedial hypothalamic nucleus on pulsatile LH release in ovariectomized rats. Neuroendocrinology 32:134 138.
Gauriau C, Bernard JF. 2002. Pain pathways and parabrachial circuits in
the rat. Exp Physiol 87:251258.
Gavioli EC, Canteras NS, De Lima TC. 1999. Anxiogenic-like effect induced by substance P injected into the lateral septal nucleus. Neuroreport 10:3399 3403.
Gorski RA, Gordon JH, Shryne JE, Southam AM. 1978. Evidence for a
morphological sex difference within the medial preoptic area of the rat
brain. Brain Res 148:333346.
Goubillon ML, Thalabard JC. 1996. Insulin-induced hypoglycemia decreases luteinizing hormone secretion in the castrated male rat: involvement of opiate peptides. Neuroendocrinology 64:49 56.
Goubillon ML, Kaufman JM, Thalabard JC. 1995. Hypothalamic multiunit
activity and pulsatile luteinizing hormone release in the castrated male
rat. Eur J Endocrinol 133:585590.
Goubillon ML, Strutton PH, OByrne KT, Thalabard JC, Coen CW. 1999.
Ketamine-induced
general
anesthesia
is
compatible
with
gonadotropin-releasing hormone pulse generator activity in gonadectomized rats: prospects for detailed electrophysiological studies in vivo.
Brain Res 841:197201.
Goubillon ML, Caraty A, Herbison AE. 2002. Evidence in favour of a direct
input from the ventromedial nucleus to gonadotropin-releasing hormone neurons in the ewe: an anterograde tracing study. J Neuroendocrinol 14:95100.
Greeley GH Jr, Nicholson GF, Nemeroff CB, Youngblood WW, Kizer JS.
1978. Direct evidence that the arcuate nucleus-median eminence tuberoinfundibular system is not of primary importance in the feedback
regulation of luteinizing hormone and follicle-stimulating hormone
secretion in the castrated rat. Endocrinology 103:170 175.
Gu GB, Simerly RB. 1997. Projections of the sexually dimorphic anteroventral periventricular nucleus in the female rat. J Comp Neurol
384:142164.
Guillamon A, Segovia S. 1997. Sex differences in the vomeronasal system.
Brain Res Bull 44:377382.
Hahn JD, Kalamatianos T, Coen CW. 2003. Studies on the neuroanatomical basis for stress-induced estrogen-potentiated suppression of reproductive function: evidence against direct corticotropin-releasing hormone projections to the vicinity of luteinizing hormone-releasing
hormone cell bodies in female rats. J Neuroendocrinol 15:732742.
Hansen S, Kohler C, Goldstein M, Steinbusch HV. 1982. Effects of ibotenic
acid-induced neuronal degeneration in the medial preoptic area and
the lateral hypothalamic area on sexual behavior in the male rat. Brain
Res 239:213232.
Helena CV, Franci CR, Anselmo-Franci JA. 2002. Luteinizing hormone
and luteinizing hormone-releasing hormone secretion is under locus
coeruleus control in female rats. Brain Res 955:245252.
Hennessey AC, Camak L, Gordon F, Edwards DA. 1990. Connections
between the pontine central gray and the ventromedial hypothalamus
are essential for lordosis in female rats. Behav Neurosci 104:477 488.
Herbert H, Moga MM, Saper CB. 1990. Connections of the parabrachial
nucleus with the nucleus of the solitary tract and the medullary reticular formation in the rat. J Comp Neurol 293:540 580.
Herbison AE. 1998. Multimodal inuence of estrogen upon gonadotropinreleasing hormone neurons. Endocr Rev 19:302330.
Herbison AE, Pape JR. 2001. New evidence for estrogen receptors in
gonadotropin-releasing hormone neurons. Front Neuroendocrinol 22:
292308.
Herbison AE, King IS, Tan KC, Dye S. 1995. Increased fos expression in
preoptic calcitonin gene-related peptide (CGRP) neurons following
mating but not the luteinizing hormone surge in female rats. J Neuroendocrinol 7:377385.
Hoffman GE, Smith MS, Verbalis JG. 1993. c-Fos and related immediate
early gene products as markers of activity in neuroendocrine systems.
Front Neuroendocrinol 14:173213.
Hoffman GE, Dohanics J, Watson RE, Wiegand SJ. 1996. The hypothalamic ventromedial nucleus sends a met-enkephalin projection to the
212
preoptic areas periventricular zone in the female rat. Brain Res Mol
Brain Res 36:201210.
Hoorneman EM, Buijs RM. 1982. Vasopressin pathways in the rat brain
following suprachiasmatic nucleus lesioning. Brain Res 243:235241.
Horvath TL, Naftolin F, Leranth C. 1992. GABAergic and catecholaminergic innervation of mediobasal hypothalamic beta-endorphin cells projecting to the medial preoptic area. Neuroscience 51:391399.
Hrabovszky E, Steinhauser A, Barabas K, Shughrue PJ, Petersen SL,
Merchenthaler I, Liposits Z. 2001. Estrogen receptor-beta immunoreactivity in luteinizing hormone-releasing hormone neurons of the rat
brain. Endocrinology 142:32613264.
Hutton LA, Gu G, Simerly RB. 1998. Development of a sexually dimorphic
projection from the bed nuclei of the stria terminalis to the anteroventral periventricular nucleus in the rat. J Neurosci 18:30033013.
Inagaki N, Toda K, Taniuchi I, Panula P, Yamatodani A, Tohyama M,
Watanabe T, Wada H. 1990. An analysis of histaminergic efferents of
the tuberomammillary nucleus to the medial preoptic area and inferior
colliculus of the rat. Exp Brain Res 80:374 380.
Jaworski RL, Piekut D, Blair ML. 2002. Pregnancy alters lateral parabrachial nucleus but not hypothalamic Fos expression following hypotensive hemorrhage. Brain Res Bull 57:595 602.
Jeong KH, Jacobson L, Widmaier EP, Majzoub JA. 1999. Normal suppression of the reproductive axis following stress in corticotropin-releasing
hormone-decient mice. Endocrinology 140:17021708.
Jurgens U. 2002. Neural pathways underlying vocal control. Neurosci
Biobehav Rev 26:235258.
Kalamatianos T, Kallo I, Goubillon ML, Coen CW. 2004a. Cellular expression of V1a vasopressin receptor mRNA in the female rat preoptic area:
effects of estrogen. J Neuroendocrinol 16:525533.
Kalamatianos T, Kallo I, Piggins.D, Coen CW. 2004b. Expression of VIP
and PACAP receptor mRNA in peptide synthesising cells within the
suprachiasmatic nucleus of the rat and in its efferent target sites.
Kallo I, Butler JA, Barkovics-Kallo M, Goubillon ML, Coen CW. 2001.
Estrogen receptor beta-immunoreactivity in gonadotropin releasing
hormone-expressing neurons: regulation by estrogen. J Neuroendocrinol 13:741748.
Kalra SP. 1993. Mandatory neuropeptide-steroid signaling for the preovulatory luteinizing hormone-releasing hormone discharge. Endocr Rev
14:507538.
Kippin TE, Cain SW, Pfaus JG. 2003. Estrous odors and sexually conditioned neutral odors activate separate neural pathways in the male rat.
Neuroscience 117:971979.
Kishore KR, Desiraju T. 1990. Inhibition of positively rewarding behavior
by the heightened aggressive state evoked either by pain-inducing
stimulus or septal lesion. Indian J Physiol Pharmacol 34:125129.
Kiss J, Halasz B. 1985. Demonstration of serotoninergic axons terminating
on luteinizing hormone-releasing hormone neurons in the preoptic area
of the rat using a combination of immunocytochemistry and high resolution autoradiography. Neuroscience 14:69 78.
Kita H, Oomura Y. 1982. An HRP study of the afferent connections to rat
medial hypothalamic region. Brain Res Bull 8:53 62.
Kitts CS, Johnson JH. 1986. Reversal by estrogen of the effect of dorsal
raphe stimulation on release of LH but not prolactin. J Neurosci Res
15:253259.
Krout KE, Jansen AS, Loewy AD. 1998. Periaqueductal gray matter projection to the parabrachial nucleus in rat. J Comp Neurol 401:437 454.
Krukoff TL, Harris KH, Jhamandas JH. 1993. Efferent projections from
the parabrachial nucleus demonstrated with the anterograde tracer
Phaseolus vulgaris leucoagglutinin. Brain Res Bull 30:163172.
Laamme N, Nappi RE, Drolet G, Labrie C, Rivest S. 1998. Expression and
neuropeptidergic characterization of estrogen receptors (ERalpha and
ERbeta) throughout the rat brain: anatomical evidence of distinct roles
of each subtype. J Neurobiol 36:357378.
Lamperti A, Blaha G. 1980. Further observations on the effects of neonatally administered monosodium glutamate on the reproductive axis of
hamsters. Biol Reprod 22:687 693.
Le WW, Berghorn KA, Rassnick S, Hoffman GE. 1999. Periventricular
preoptic area neurons coactivated with luteinizing hormone (LH)releasing hormone (LHRH) neurons at the time of the LH surge are
LHRH afferents. Endocrinology 140:510 519.
Le WW, Wise PM, Murphy AZ, Coolen LM, Hoffman GE. 2001. Parallel
declines in fos activation of the medial anteroventral periventricular
nucleus and lhrh neurons in middle-aged rats. Endocrinology 142:
4976 4982.

Leak RK, Moore RY. 2001. Topographic organization of suprachiasmatic
nucleus projection neurons. J Comp Neurol 433:312334.
Leanza G, Pellitteri R, Russo A, Stanzani S. 1989. Branching projections
from subcoeruleus area neurons to medial preoptic area and cervical
spinal cord revealed by double retrograde neuronal labeling. Neurosci
Lett 103:1116.
Leanza G, Pellitteri R, Russo A, Stanzani S. 1991. Neurons in raphe nuclei
pontis and magnus have branching axons that project to medial preoptic area and cervical spinal cord. A uorescent retrograde double
labeling study in the rat. Neurosci Lett 123:195199.
Lee WS, Smith MS, Hoffman GE. 1990. Luteinizing hormone-releasing
hormone neurons express Fos protein during the prestrous surge of
luteinizing hormone. Proc Natl Acad Sci U S A 87:51635167.
Levine JE, Bauer-Dantoin AC, Besecke LM, Conaghan LA, Legan SJ,
Meredith JM, Strobl FJ, Urban JH, Vogelsong KM, Wolfe AM. 1991.
Neuroendocrine regulation of the luteinizing hormone-releasing hormone pulse generator in the rat. Rec Prog Horm Res 47:97151.
Li HY, Sawchenko PE. 1998. Hypothalamic effector neurons and extended
circuitries activated in neurogenic stress: a comparison of footshock
effects exerted acutely, chronically, and in animals with controlled
glucocorticoid levels. J Comp Neurol 393:244 266.
Li X, Schwartz PE, Rissman EF. 1997. Distribution of estrogen receptorbeta-like immunoreactivity in rat forebrain. Neuroendocrinology 66:
63 67.
Li XF, Mitchell JC, Wood S, Coen CW, Lightman SL, OByrne KT. 2003.
The effect of estradiol and progesterone on hypoglycaemic stressinduced suppression of pulsatile luteinizing hormone release and on
corticotropin-releasing hormone mRNA expression in the rat. J Neuroendocrinol 15:468 470.
Limonta P, Maggi R, Giudici D, Martini L, Piva F. 1981. Role of the
subfornical organ (SFO) in the control of gonadotropin secretion. Brain
Res 229:75 84.
Lin SH, Miyata S, Matsunaga W, Kawarabayashi T, Nakashima T, Kiyohara T. 1998. Metabolic mapping of the brain in pregnant, parturient
and lactating rats using fos immunohistochemistry. Brain Res 787:
226 236.
Lu H, Ozawa H, Nishi M, Ito T, Kawata M. 2001. Serotonergic neurons in
the dorsal raphe nucleus that project into the medial preoptic area
contain estrogen receptor beta. J Neuroendocrinol 13:839 845.
Luppi PH, Aston-Jones G, Akaoka H, Chouvet G, Jouvet M. 1995. Afferent
projections to the rat locus coeruleus demonstrated by retrograde and
anterograde tracing with cholera-toxin B subunit and Phaseolus vulgaris leucoagglutinin. Neuroscience 65:119 160.
MacDonald MC, Wilkinson M. 1990. Peripubertal treatment with
N-methyl-D-aspartic acid or neonatally with monosodium glutamate
accelerates sexual maturation in female rats, an effect reversed by
MK-801. Neuroendocrinology 52:143149.
Maeda K, Cagampang FR, Coen CW, Tsukamura H. 1994. Involvement of
the catecholaminergic input to the paraventricular nucleus and of
corticotropin-releasing hormone in the fasting-induced suppression of
luteinizing hormone release in female rats. Endocrinology 134:1718
1722.
Malpaux B, Daveau A, Maurice-Mandon F, Duarte G, Chemineau P. 1998.
Evidence that melatonin acts in the premammillary hypothalamic area
to control reproduction in the ewe: presence of binding sites and stimulation of luteinizing hormone secretion by in situ microimplant delivery. Endocrinology 139:1508 1516.
Mani SK, Allen JM, Rettori V, McCann SM, OMalley BW, Clark JH. 1994.
Nitric oxide mediates sexual behavior in female rats. Proc Natl Acad
Sci U S A 91:6468 6472.
Martins-Afferri MP, Ferreira-Silva IA, Franci CR, Anselmo-Franci JA.
2003. LHRH release depends on locus coeruleus noradrenergic inputs
to the medial preoptic area and median eminence. Brain Res Bull
61:521527.
McGinty DJ, Sterman MB. 1968. Sleep suppression after basal forebrain
lesions in the cat. Science 160:12531255.
Merchenthaler I, Gorcs T, Setalo G, Petrusz P, Flerko B. 1984.
Gonadotropin-releasing hormone (GnRH) neurons and pathways in the
rat brain. Cell Tissue Res 237:1529.
Mihaly E, Legradi G, Fekete C, Lechan RM. 2001. Efferent projections of
ProTRH neurons in the ventrolateral periaqueductal gray. Brain Res
919:185197.
Mizusawa A, Ogawa H, Kikuchi Y, Hida W, Shirato K. 1995. Role of the
parabrachial nucleus in ventilatory responses of awake rats. J Physiol
489:877 884.

Moga MM, Gray TS. 1985. Evidence for corticotropin-releasing factor,
neurotensin, and somatostatin in the neural pathway from the central
nucleus of the amygdala to the parabrachial nucleus. J Comp Neurol
241:275284.
Moga MM, Saper CB, Gray TS. 1989. Bed nucleus of the stria terminalis:
cytoarchitecture, immunohistochemistry, and projection to the parabrachial nucleus in the rat. J Comp Neurol 283:315332.
Moga MM, Herbert H, Hurley KM, Yasui Y, Gray TS, Saper CB. 1990.
Organization of cortical, basal forebrain, and hypothalamic afferents to
the parabrachial nucleus in the rat. J Comp Neurol 295:624 661.
Moore RY, Speh JC, Leak RK. 2002. Suprachiasmatic nucleus organization. Cell Tissue Res 309:89 98.
Morello H, Taleisnik S. 1985. Changes of the release of luteinizing hormone
(LH) on the day of prestrus after lesions or stimulation of the raphe
nuclei in rats. Brain Res 360:311317.
Morello H, Taleisnik S. 1988. The inhibition of prestrous LH surge and
ovulation in rats bearing lesions of the dorsal raphe nucleus is mediated by the locus coeruleus. Brain Res 440:227231.
Moss RL, McCann SM. 1973. Induction of mating behavior in rats by
luteinizing hormone-releasing factor. Science 181:177179.
Mufson EJ, Cai WJ, Jaffar S, Chen E, Stebbins G, Sendera T, Kordower
JH. 1999. Estrogen receptor immunoreactivity within subregions of the
rat forebrain: neuronal distribution and association with perikarya
containing choline acetyltransferase. Brain Res 849:253274.
Nagatani S, Bucholtz DC, Murahashi K, Estacio MA, Tsukamura H, Foster
DL, Maeda KI. 1996. Reduction of glucose availability suppresses pulsatile luteinizing hormone release in female and male rats. Endocrinology 137:1166 1170.
Numan M, Numan M. 1996. A lesion and neuroanatomical tract-tracing
analysis of the role of the bed nucleus of the stria terminalis in retrieval
behavior and other aspects of maternal responsiveness in rats. Dev
Psychobiol 29:2351.
Olney JW. 1969. Brain lesions, obesity, and other disturbances in mice
treated with monosodium glutamate. Science 164:719 721.
Ono N, Lumpkin MD, Samson WK, McDonald JK, McCann SM. 1984.
Intrahypothalamic action of corticotrophin-releasing factor (CRF) to
inhibit growth hormone and LH release in the rat. Life Sci 35:1117
1123.
Orikasa C, Sakuma Y. 2003. Possible involvement of preoptic estrogen
receptor beta positive cells in luteinizing hormone surge in the rat.
Domest Anim Endocrinol 25:8392.
Orikasa C, Kondo Y, Hayashi S, McEwen BS, Sakuma Y. 2002. Sexually
dimorphic expression of estrogen receptor beta in the anteroventral
periventricular nucleus of the rat preoptic area: implication in luteinizing hormone surge. Proc Natl Acad Sci U S A 99:3306 3311.
Palm IF, van der Beek EM, Wiegant VM, Buijs RM, Kalsbeek A. 1999.
Vasopressin induces a luteinizing hormone surge in ovariectomized,
estradiol-treated rats with lesions of the suprachiasmatic nucleus.
Neuroscience 93:659 666.
Palm IF, van der Beek EM, Wiegant VM, Buijs RM, Kalsbeek A. 2001. The
stimulatory effect of vasopressin on the luteinizing hormone surge in
ovariectomized, estradiol-treated rats is time-dependent. Brain Res
901:109 116.
Paxinos G, Watson C. 1998. The rat brain in stereotaxic coordinates. San
Diego: Academic Press.
Perez-Clausell J, Frederickson CJ, Danscher G. 1989. Amygdaloid efferents through the stria terminalis in the rat give origin to zinccontaining boutons. J Comp Neurol 290:201212.
Peter Z, Churchill L, Hajdu I, Obal JF, Krueger JM, Parducz A. 2004.
Fos-immunoreactivity in the hypothalamus: dependency on the diurnal
rhythm, sleep, gender, and estrogen. Neuroscience 124:695707.
Petersen SL, McCrone S, Keller M, Shores S. 1995. Effects of estrogen and
progesterone on luteinizing hormone-releasing hormone messenger ribonucleic acid levels: consideration of temporal and neuroanatomical
variables. Endocrinology 136:3604 3610.
Petersen SL, Ottem EN, Carpenter CD. 2003. Direct and indirect regulation of gonadotropin-releasing hormone neurons by estradiol. Biol Reprod 69:17711778.
Petraglia F, Sutton S, Vale W, Plotsky P. 1987. Corticotropin-releasing
factor decreases plasma luteinizing hormone levels in female rats by
inhibiting gonadotropin-releasing hormone release into hypophysialportal circulation. Endocrinology 120:10831088.
Petrov T, Krukoff TL, Jhamandas JH. 1992. The hypothalamic paraventricular and lateral parabrachial nuclei receive collaterals from raphe
213
nucleus neurons: a combined double retrograde and immunocytochemical study. J Comp Neurol 318:18 26.
Pfaff DW. 1973. Luteinizing hormone-releasing factor potentiates lordosis
behavior in hypophysectomized ovariectomized female rats. Science
182:1148 1149.
Pfaff DW, Sakuma Y. 1979a. Decit in the lordosis reex of female rats
caused by lesions in the ventromedial nucleus of the hypothalamus.
J Physiol 288:203210.
Pfaff DW, Sakuma Y. 1979b. Facilitation of the lordosis reex of female
rats from the ventromedial nucleus of the hypothalamus. J Physiol
288:189 202.
Pfaff DW, Schwartz-Giblin S, McCarthy MM, Kow L. 1994. Cellular mechanisms of female reproductive behaviours. In: Knobil E, Neill JD,
editors. The physiology of reproduction. New York: Raven. p 107220.
Pi X, Voogt JL. 2000. Effect of suckling on prolactin receptor immunoreactivity in the hypothalamus of the rat. Neuroendocrinology 71:308
317.
Polston EK, Gu G, Simerly RB. 2004. Neurons in the principal nucleus of
the bed nuclei of the stria terminalis provide a sexually dimorphic
GABAergic input to the anteroventral periventricular nucleus of the
hypothalamus. Neuroscience 123:793 803.
Porkka-Heiskanen T, Urban JH, Turek FW, Levine JE. 1994. Gene expression in a subpopulation of luteinizing hormone-releasing hormone
(LHRH) neurons prior to the preovulatory gonadotropin surge. J Neurosci 14:5548 5558.
Rajendren G, Moss RL. 1993. The role of the medial nucleus of amygdala
in the mating-induced enhancement of lordosis in female rats: the
interaction with luteinizing hormone-releasing hormone neuronal system. Brain Res 617:81 86.
Reilly S. 1999. The parabrachial nucleus and conditioned taste aversion.
Brain Res Bull 48:239 254.
Riskind P, Moss RL. 1983. Midbrain LHRH infusions enhance lordotic
behavior in ovariectomized estrogen-primed rats independently of a
hypothalamic responsiveness to LHRH. Brain Res Bull 11:481 485.
Rivest S, Rivier C. 1991. Inuence of the paraventricular nucleus of the
hypothalamus in the alteration of neuroendocrine functions induced by
intermittent footshock or interleukin. Endocrinology 129:2049 2057.
Rivier C, Vale W. 1984. Inuence of corticotropin-releasing factor on reproductive functions in the rat. Endocrinology 114:914 921.
Rivier C, Rivier J, Vale W. 1986. Stress-induced inhibition of reproductive
functions: role of endogenous corticotropin-releasing factor. Science
231:607 609.
Ronnekleiv OK, Kelly MJ. 1986. Luteinizing hormone-releasing hormone
neuronal system during the estrous cycle of the female rat: effects of
surgically induced persistent estrus. Neuroendocrinology 43:564 576.
Ronnekleiv OK, Kelly MJ. 1988. Plasma prolactin and luteinizing hormone
proles during the estrous cycle of the female rat: effects of surgically
induced persistent estrus. Neuroendocrinology 47:133141.
Roozendaal MM, Swarts JJ, Wolbers WB, Threels A, Wiegant VM, Mattheij JA. 1996. Effect of CRH on the preovulatory LH and FSH surge in
the cyclic rat: a role for arginine vasopressin? J Neuroendocrinol 8:765
770.
Sakuma Y, Pfaff DW. 1979. Facilitation of female reproductive behavior
from mesencephalic central gray in the rat. Am J Physiol 237:R278
R284.
Sakumoto T, Tohyama M, Satoh K, Kimoto Y, Kinugasa T, Tanizawa O,
Kurachi K, Saper CB, Swanson LW, Cowan WM. 1976. The efferent
connections of the ventromedial nucleus of the hypothalamus of the rat.
Schutterbrandt TG, David, JT, Szabo F, Hoffman GE. 2004. Subregions of
the VMN involved in neuroendocrine control are larger in females than
in males. Program No. 885.3, Abstract Viewer/Itinerary Planner.
Washington, DC: Society for Neuroscience.
Schwanzel-Fukuda M, Bick D, Pfaff DW. 1989. Luteinizing hormonereleasing hormone (LHRH)-expressing cells do not migrate normally in
an inherited hypogonadal (Kallmann) syndrome. Brain Res Mol Brain
Res 6:311326.
Scott CJ, Rawson JA, Pereira AM, Clarke IJ. 1999. Estrogen receptors in
the brainstem of the female sheep: relationship to noradrenergic cells
and cells projecting to the medial preoptic area. J Neuroendocrinol
11:745755.
Sheehan T, Paul M, Amaral E, Numan MJ, Numan M. 2001. Evidence that
the medial amygdala projects to the anterior/ventromedial hypothalamic nuclei to inhibit maternal behavior in rats. Neuroscience 106:
341356.
214
Shimizu N. 1978. Afferent ber connections from lower brain stem to
hypothalamus studied by the horseradish peroxidase method with
special reference to noradrenaline innervation. Exp Brain Res 31:81
94.
Shughrue PJ, Merchenthaler I. 2001. Distribution of estrogen receptor
beta immunoreactivity in the rat central nervous system. J Comp
Neurol 436:64 81.
Shughrue PJ, Lane MV, Merchenthaler I. 1997. Comparative distribution
of estrogen receptor-alpha and -beta mRNA in the rat central nervous
system. J Comp Neurol 388:507525.
Simerly RB, Swanson LW. 1986. The organization of neural inputs to the
medial preoptic nucleus of the rat. J Comp Neurol 246:312342.
Simerly RB, Swanson LW. 1988. Projections of the medial preoptic nucleus: a Phaseolus vulgaris leucoagglutinin anterograde tract-tracing
study in the rat. J Comp Neurol 270:209 242.
Simerly RB, Chang C, Muramatsu M, Swanson LW. 1990. Distribution of
androgen and estrogen receptor mRNA-containing cells in the rat
brain: an in situ hybridization study. J Comp Neurol 294:76 95.
Simonian SX, Spratt DP, Herbison AE. 1999. Identication and characterization of estrogen receptor alpha-containing neurons projecting to the
vicinity of the gonadotropin-releasing hormone perikarya in the rostral
preoptic area of the rat. J Comp Neurol 411:346 358.
Spector AC. 1995. Gustatory function in the parabrachial nuclei: implications from lesion studies in rats. Rev Neurosci 6:143175.
Sridaran R, Rodriguez-Sierra JF, Blake CA. 1981. Effects of hypothalamic
arcuate nucleus lesions on pulsatile luteinizing hormone concentration
in ovariectomized rats. Proc Soc Exp Biol Med 168:38 44.
Sumida H, Nishizuka M, Kano Y, Arai Y. 1993. Sex differences in the
anteroventral periventricular nucleus of the preoptic area and in the
related effects of androgen in prenatal rats. Neurosci Lett 151:41 44.
Swanson LW. 1995. Mapping the human brain: past, present, and future.
Trends Neurosci 18:471 474.
Szymusiak R, Satinoff E. 1984. Ambient temperature-dependence of sleep
disturbances produced by basal forebrain damage in rats. Brain Res
Bull 12:295305.
Takano S, Negoro H, Honda K, Higuchi T. 1992. Lesion and electrophysiological studies on the hypothalamic afferent pathway of the milk
ejection reex in the rat. Neuroscience 50:877 883.
Terasawa E, Wiegand SJ, Bridson WE. 1980. A role for medial preoptic
nucleus on afternoon of prestrus in female rats. Am J Physiol 238:
E533E539.
Thompson RH, Swanson LW. 2003. Structural characterization of a hypothalamic visceromotor pattern generator network. Brain Res Brain Res
Rev 41:153202.
Tillet Y. 1992. Serotoninergic projections from the raphe nuclei to the
preoptic area in sheep as revealed by immunohistochemistry and retrograde labeling. J Comp Neurol 320:267272.
Tillet Y, Batailler M, Thibault J. 1993. Neuronal projections to the medial
preoptic area of the sheep, with special reference to monoaminergic
afferents: immunohistochemical and retrograde tract tracing studies.
J Comp Neurol 330:195220.
Tsukahara S, Tsukamura H, Foster DL, Maeda KI. 1999. Effect of
corticotropin-releasing hormone antagonist on estrogen-dependent glucoprivic suppression of luteinizing hormone secretion in female rats.
J Neuroendocrinol 11:101105.
Van der Beek EM, Wiegant VM, van der Donk HA, Van den Hurk R, Buijs
RM. 1993. Lesions of the suprachiasmatic nucleus indicate the pres-

ence of a direct vasoactive intestinal polypeptide-containing projection
to gonadotrophin-releasing hormone neurons in the female rat. J Neuroendocrinol 5:137144.
Van der Beek EM, Horvath TL, Wiegant VM, Van den Hurk R, Buijs RM.
1997. Evidence for a direct neuronal pathway from the suprachiasmatic nucleus to the gonadotropin-releasing hormone system: combined tracing and light and electron microscopic immunocytochemical
studies. J Comp Neurol 384:569 579.
Vertes RP, Kocsis B. 1994. Projections of the dorsal raphe nucleus to the
brainstem: PHA-L analysis in the rat. J Comp Neurol 340:1126.
Wakerley JB, Cosgrave AS. 2002. Ventral tegmental lesions disrupt the
facilitatory effect of central oxytocin on the milk-ejection reex. 5th
International Conference of Neuroendocrinology Abstracts, p 275.
Watson RE, Wiegand SJ, Clough RW, Hoffman GE. 1986. Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue
morphology. Peptides 7:155159.
Watson RE, Langub MC, Engle MG, Maley BE. 1995. Estrogen-receptive
neurons in the anteroventral periventricular nucleus are synaptic targets of the suprachiasmatic nucleus and peri-suprachiasmatic region.
Brain Res 689:254 264.
Watts AG, Swanson LW, Sanchez-Watts G. 1987. Efferent projections of
the suprachiasmatic nucleus: I. Studies using anterograde transport of
Phaseolus vulgaris leucoagglutinin in the rat. J Comp Neurol 258:204
229.
Wedemeyer C, Vivas L, Carrer HF. 1999. Peripeduncular lesion alters
expression of c-fos induced in the female rat brain by male mounting.
J Neuroendocrinol 11:221228.
Wiegand SJ, Terasawa E. 1982. Discrete lesions reveal functional heterogeneity of suprachiasmatic structures in regulation of gonadotropin
secretion in the female rat. Neuroendocrinology 34:395 404.
Wiegand SJ, Terasawa E, Bridson WE. 1978. Persistent estrus and blockade of progesterone-induced LH release follows lesions which do not
damage the suprachiasmatic nucleus. Endocrinology 102:16451648.
Wiegand SJ, Terasawa E, Bridson WE, Goy RW. 1980. Effects of discrete
lesions of preoptic and suprachiasmatic structures in the female rat.
Alterations in the feedback regulation of gonadotropin secretion. Neuroendocrinology 31:147157.
Witkin JW, Paden CM, Silverman AJ. 1982. The luteinizing hormonereleasing hormone (LHRH) systems in the rat brain. Neuroendocrinology 35:429 438.
Wray S, Hoffman G. 1986. A developmental study of the quantitative
distribution of LHRH neurons within the central nervous system of
postnatal male and female rats. J Comp Neurol 252:522531.
Wright DE, Jennes L. 1993. Origin of noradrenergic projections to GnRH
perikarya-containing areas in the medial septum-diagonal band and
preoptic area. Brain Res 621:272278.
Yokosuka M, Okamura H, Hayashi S. 1997. Postnatal development and
sex difference in neurons containing estrogen receptor-alpha immunoreactivity in the preoptic brain, the diencephalon, and the amygdala in
the rat. J Comp Neurol 389:8193.
Zardetto-Smith AM, Gray TS. 1987. A direct neural projection from the
nucleus of the solitary tract to the subfornical organ in the rat. Neurosci Lett 80:163166.
Zhang JQ, Cai WQ, Zhou DS, Su BY. 2002. Distribution and differences of
estrogen receptor beta immunoreactivity in the brain of adult male and
female rats. Brain Res 935:73 80.

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THE JOURNAL OF COMPARATIVE NEUROLOGY 494:190 214 (2006)

Comparative Study of the Sources of

The neurons that synthesize gonadotrophin-releasing

1980; Wiegand and Terasawa, 1982; Ronnekleiv and

Grant sponsor: Biotechnology and Biological Sciences Research Council

medial amygdaloid nucleus, posteroventral part

J.D. HAHN AND C.W. COEN

MATERIALS AND METHODS

RETROGRADE TRACING FROM GnRH CELL SITE OR AVPV

fate hexahydrate. The reaction was monitored under a

Control procedures for ICC

J.D. HAHN AND C.W. COEN

quently in photoimaging software (Adobe Photoshop

RETROGRADE TRACING FROM GnRH CELL SITE OR AVPV

J.D. HAHN AND C.W. COEN

sparsely labelled from either site. Retrograde labelling of

Mesencephalon and rhombencephalon

RETROGRADE TRACING FROM GnRH CELL SITE OR AVPV

the GnRH perikarya site or the AVPV was most abundant

Fig. 2. Brain maps in the coronal plane showing the distribution

RETROGRADE TRACING FROM GnRH CELL SITE OR AVPV

J.D. HAHN AND C.W. COEN

RETROGRADE TRACING FROM GnRH CELL SITE OR AVPV

J.D. HAHN AND C.W. COEN

Fig. 3. Summary diagram illustrating the sites and relative

the possibility that it participates in relaying autonomic

and Simerly, 1997; Le et al., 1999, 2001; Simonian et al.,

RETROGRADE TRACING FROM GnRH CELL SITE OR AVPV

In female hamsters, SCN efferents have been observed

J.D. HAHN AND C.W. COEN

RETROGRADE TRACING FROM GnRH CELL SITE OR AVPV

Fig. 4. Representative brighteld photomicrographs showing

J.D. HAHN AND C.W. COEN

RETROGRADE TRACING FROM GnRH CELL SITE OR AVPV

glutamate; these effects include reduced ovarian weight

J.D. HAHN AND C.W. COEN

Mesencephalon and rhombencephalon

RETROGRADE TRACING FROM GnRH CELL SITE OR AVPV

fasting in ovariectomized, estrogen-replaced rats (Cagampang et al., 1992).

J.D. HAHN AND C.W. COEN

RETROGRADE TRACING FROM GnRH CELL SITE OR AVPV

attenuates the surge of luteinizing hormone and prolactin secretion in

J.D. HAHN AND C.W. COEN

RETROGRADE TRACING FROM GnRH CELL SITE OR AVPV

J.D. HAHN AND C.W. COEN

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