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 Previous Article
The Journal of Neuroscience, April 1, 2001, 21(7):2546-2552
CNS Region-Specific Oxytocin Receptor Expression: Importance in
Regulation of Anxiety and Sex Behavior
Tracy L.
Bale1,
Aline
M.
Davis2,
Anthony P.
Auger2,
Daniel M.
Dorsa1, and
Margaret M.
McCarthy2
1 Neurobiology and Behavior Program, Departments of
Pharmacology and Psychiatry, University of Washington, Seattle,
Washington 98195, and 2 Department of Physiology,
University of Maryland, Baltimore, Maryland 21201
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ABSTRACT |
The oxytocin receptor (OTR) is differentially expressed in the CNS.
Because there are multiple mechanisms by which the OTR can be
transcriptionally induced, we hypothesized that differences in OTR
expression may be explained by activation of distinct signal transduction pathways and may be critical for the control of anxiety and sex behaviors. To determine the regulation and functional significance of this expression, we infused female rats with modifiers of protein kinases before assaying for behavior and oxytocin receptor binding. In the ventromedial nucleus of the hypothalamus (VMH), estrogen-dependent induction of oxytocin receptors required protein kinase C activation, and oxytocin infused here promoted female sex
behavior but had no effect on anxiety. In contrast, dopamine controlled
tonic oxytocin receptor expression in the central nucleus of the
amygdala (cAmyg) through activation of protein kinase A, and oxytocin
infused here was anxiolytic but had no effect on female sex behavior.
Therefore, we have identified brain region-specific regulation of the
OTR in the VMH and cAmyg. Distinct signal transduction pathways
regulating receptor expression and binding in each brain region may
mediate in part the ability of oxytocin to exert these differential
behavioral effects.
Key words:
oxytocin; heterologous expression; amygdala; hypothalamus; lordosis; anxiety
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INTRODUCTION |
The neuropeptide oxytocin (OT) is
synthesized in neurons of the supraoptic and paraventricular
(PVN) nuclei of the hypothalamus, which project to the posterior
pituitary and release oxytocin into the bloodstream. This peripherally
acting oxytocin is critical for the processes of lactation and
parturition. Oxytocin neurons of the PVN also send projections to many
regions within the brain, including the hippocampus, amygdala, and
hypothalamus, in which oxytocin acts as a neurotransmitter involved in
affiliative behaviors (Insel, 1992 ). Oxytocin receptors (OTRs) are
found in many brain regions, including the central nucleus of the
amygdala (cAmyg) and the ventromedial nucleus of the hypothalamus (VMH)
in which they are heterologously expressed (Bale et al.,
1995a,b). These two brain regions are critical components of
neural circuits regulating distinct behavioral responses. The VMH is an
important mediator of female sex behavior (Pfaff et al., 1994), whereas
the cAmyg is a portion of the limbic system that coordinates fear and
anxiety responses (Davis, 1997; Roozendaal et al., 1997; LeDoux, 1998). The exhibition of rat female sex behavior requires estrogen action in
the brain, including the induction of OTRs in the VMH (McCarthy et al.,
1994; Bale and Dorsa, 1995a,b; Bale et al., 1995a,b). In
contrast, OTR levels in the cAmyg are unaffected by estradiol (Bale and
Dorsa, 1995a), despite dense collections of estrogen-concentrating neurons (Pfaff and Keiner, 1973).
In vitro, OTR expression is increased by activation of
protein kinase A (PKA) or protein kinase C (PKC) (Bale and Dorsa,
1998). Previously, we isolated and sequenced 4.5 kb of an upstream
sequence of a rat genomic OTR clone from a rat testis library and
identified crucial putative response elements, including a cAMP
response element and several activator protein-1 (AP-1) sites (Bale and Dorsa, 1997). Because there are multiple mechanisms by which the OTR
can be induced transcriptionally, we hypothesized that differences in
OTR expression between the VMH and cAmyg may be explained by activation
of distinct signal transduction pathways in these brain regions, and
these may be critical for the control of anxiety and sex behaviors.
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MATERIALS AND METHODS |
Animals. Sprague Dawley female rats were obtained
from Charles River (Kingston, NY) and housed under reversed light/dark
cycle conditions (lights out at 10:00 A.M.) in the animal facility of the University of Maryland, Baltimore. All animals were ovariectomized under anesthesia [75 mg of ketamine + 2.5 mg of acepromazine/kg body
weight (BW)] and allowed at least 1 week to recover before the
experiment. All procedures were in accordance with the guidelines of
the University of Maryland Institutional Animal Care and Use Committee.
Acute intracerebral infusions for oxytocin receptor binding.
Animals were anesthetized (75 mg of ketamine + 2.5 mg of
acepromazine/kg BW) and placed into a stereotaxic apparatus for
site-specific infusions using a 1 µl Hamilton syringe with attached
needle. Stereotaxic coordinates for infusion into the amygdala were 2.8 mm caudal to bregma, 3.75 mm lateral to the midline, and 8.00 mm below
the surface of the brain. Coordinates for injections into the VMH were
the same, except 0.75 mm lateral to the midline. These coordinates were
based on Figure 29 of the Paxinos and Watson (1986) rat brain atlas.
Bilateral infusions (1 µl per side) consisted of phorbol ester (200 nM; Sigma, St. Louis, MO), forskolin (10 µM; Sigma), H89 (200 µM; Calbiochem, La Jolla CA),
bisindolylmalemide I (BIM, 200 µM; Calbiochem),
or the dopamine D1 receptor antagonist R(+)-SCH-23390
hydrochloride (10 µg; Research Biochemicals, Natick, MA).
Infusions were ~1 min in duration, and the injection cannula was left
in place for an additional 30 sec before removal. At completion of the
infusion, the incision was closed with surgical staples; the animal was
treated with antibiotics and an analgesic and injected subcutaneously
with either 2 µg of estradiol benzoate (EB) or oil vehicle in 0.1 cc
of sesame oil before being returned to its home cage. To evaluate the
inhibitory effect of BIM on OTR expression in the VMH, EB treatment is
necessary to induce a detectable level of OTRs in this brain region. No
EB treatment is necessary for OTR expression in the cAmyg because
endogenous levels are maximal without estrogen exposure, and therefore
ovariectomized females were left untreated.
Oxytocin receptor binding. Eight hours after infusion,
animals were anesthetized lightly with CO2 before
decapitation. Brains were removed rapidly, frozen briefly in
isopentane, and stored at  80°C until sectioning. Brains were cut on
a cryostat (20 µm thick), and sections were mounted onto subbed
slides and frozen at 80°C until binding was performed. Slides were
thawed before being washed twice for 10 min in 50 mM Tris buffer, pH 7.4, at room temperature.
Sections were then incubated in buffer containing: 50 mM Tris, pH 7.4, 30 pM
125I-Ornithine Vasotocin (OTA; DuPont NEN,
Boston, MA), 10 mM
MgCl2, 0.1% BSA, and 0.05% bacitracin for 60 min at room temperature. Nonspecific binding was defined in adjacent
sections that were exposed to both
125I-OTA and 1 µM
cold OT (Bachem, Torrance, CA). After the incubation period,
slides were washed four times for 5 min each in a 50 mM Tris buffer containing 10 mM MgCl2 at 4°C. Slides
were dipped rapidly in deionized water and dried under cool air.
Sections were then exposed to film for 3 d before being developed
by standard photographic methods.
Autoradiography data analysis. Film autoradiographs were
analyzed blind using a microcomputer-based image analysis system (MCID;
Image Research, St. Catherines, Ontario, Canada). Oxytocin receptor
binding optical density from autoradiographs was measured by
subtracting nonspecific signal of adjacent sections from total signal
to obtain the specific signal for each tissue section. The area of
measurement for all samples was identical and was taken from the same
region of the VMH or cAmyg as determined by choosing matching brain
sections before analysis.
Repeated intracerebral infusions for behavioral testing.
Animals were anesthetized (75 mg of ketamine + 2.5 mg of
acepromazine/kg BW), placed into a stereotaxic apparatus, and implanted
with a bilateral guide cannula (22 gauge; Plastics One, Roanoke, VA) into either the VMH or cAmyg using the same stereotaxic coordinates as
for the acute infusions. The guide cannula extended 7.5 mm into the
brain, and the internal dummy cannula extended an additional 0.5 mm.
Animals were ovariectomized at the same time as the implantation of the
cannula. At the time of infusion, awake animals were lightly restrained
in a terry cloth towel, the dummy cannula was removed, and an injection
cannula attached to a 1 µl Hamilton syringe via PE-10 tubing was
inserted into the guide cannula. After infusions were performed in the
same manner as described above, the fully awake animal was returned to
its home cage.
Depending on the experiment, animals were infused with either the D1
antagonist SCH23390 (10 µg), the PKC inhibitor BIM (200 µM), or the PKC activator phorbol ester (PMA, 200 nM) in the morning (~9:00 A.M.) and the evening (~5:30
P.M.) on 2 consecutive days and again on the morning of the third day,
for a total of five infusions. On the afternoon of the third day (the
midpoint of the dark cycle), animals were infused with either oxytocin
(1 µg) or saline vehicle 10-20 min before behavioral testing.
Female sex behavior testing. Ovariectomized females were
treated with EB (in sesame oil vehicle, 2-5 µg/0.1 cc, s.c.)
48 and 24 hr before behavioral testing. At the midpoint of the dark
cycle on the day of the test, animals were allowed to acclimate to a test arena before being tested manually for lordosis responsiveness by
an investigator blinded to the particular treatment. Testing consisted
of 10 trials of gently palpating the flanks and exerting pressure on
the rump to induce a lordosis response. The degree of arching of the
back and elevation of the rump was an indicator of the amplitude of the
response and was assigned qualitative scores from zero (no lordosis) to
three (maximal lordosis). Animals were pretested, infused with either
oxytocin (1 µg) or saline vehicle, and post-tested within 10-20 min
of infusion. Because animal variability prevents absolute numbers for
lordosis response, results were calculated as the difference for each
animal between pretest and post-test scores. For this and all other
behavioral tests, the experimenter scoring the response was blinded to
the experimental group. All animals were treated and tested at the same
time by the same investigators.
Open-field testing. At the midpoint of the dark phase of the
cycle, animals were placed for 5 min in the center of an open-field apparatus made of clear Plexiglas (100 cm long, 80 cm wide, and 50 cm
high); the floor of the apparatus was divided into 30 equally sized
squares ~16.5 cm per side. The movement of the animal was monitored
such that each crossing over a line in the grid was noted. The squares
around the perimeter were considered "outside," whereas all others
were considered "inside." Data were expressed as the ratio of
inside grid crossings to outside grid crossings. Because this was a
test of novelty-induced anxiety, animals could not be pretested before
drug treatment. Animals were tested in the apparatus 10-20 min after
an infusion of oxytocin (1 µg) or saline. An increase in the inside
crossings/outside crossings ratio is an indicator of decreased
anxiety. Animals tested for anxiety after infusion into the
cAmyg were not pretreated with EB because OTRs in this brain region are
maximal without estrogen exposure.
Elevated plus maze testing. Immediately after the open-field
test, animals were placed in the center of a standard elevated plus
maze (EPM) apparatus of 50 cm height with 50-cm-long arms arranged
perpendicularly. Two opposite arms were open, and two were enclosed
with darkened Plexiglas walls. A 40 W bulb was suspended directly above
the maze to provide even illumination to all arms. The number of
entries and time spent in open and closed arms of the maze were
recorded for 10 min with a digital recorder.
Histology. At the completion of the behavioral experiments,
all animals were overdosed with Nembutal and perfused transcardially with ice-cold saline followed by 4% paraformaldehyde. Brains were removed, post-fixed for 1 hr, cryoprotected in 30% sucrose, and stored
frozen at 80°C until sectioned on a cryostat. Sections (50 µm
thick) were collected throughout the area of the cannula implant and
stained with cresyl violet for verification of correct placement.
Statistics. Binding data and lordosis responding were
assessed for statistical significance by ANOVA, and individual
comparisons were made by post hoc Kruskal-Wallis test.
Open-field data expressed as a ratio were assessed by the nonparametric
Mann-Whitney U test.
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RESULTS |
Experiment I: Assessment of OTR binding by receptor autoradiography
after manipulation of PKA or PKC by infusion into the VMH or cAmyg
Ventromedial hypothalamus
Representative autoradiographs of oxytocin binding in the VMH
after the various treatments are pictured in Figure
1. Quantification of binding showed that
infusion of the PKC inhibitor, BIM (200 µM),
significantly decreased OTR binding levels in the VMH compared with
saline vehicle-infused, ovariectomized estradiol-replaced females (ANOVA; p < 0.01; n = 4) (Fig. 2A). Infusion
of a phorbol ester (PMA, 200 nM) to mimic
activation of PKC induced a significant increase in OTR binding in the
absence of estrogen, compared with saline vehicle-infused
ovariectomized controls (p < 0.001;
n = 4-5) (Fig. 2A). Infusion into
the VMH of H89 (200 µM; n = 3)
to inhibit PKA in ovariectomized estradiol-replaced females had no effect on OTR binding, nor did infusion of forskolin (200 nM; n = 3) to enhance PKA in
ovariectomized females (Fig. 2B)
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Figure 1.
Representative autoradiographs illustrating
oxytocin receptor binding in the VMH. Animals were pretreated with
estradiol (2 µg) or oil vehicle by subcutaneous injection; infused
centrally, directly into the VMH with BIM (inhibitor of PKC) or the
phorbol ester, PMA, 8 hr before collection of the brains; and assayed
for OTR binding by quantitative receptor autoradiography.
Treatment groups were as follows: A, subcutaneous oil
vehicle injection, saline vehicle infusion into the VMH;
B, subcutaneous estradiol injection, saline vehicle
infusion into the VMH; C, subcutaneous estradiol
injection, BIM infusion into the VMH; D, subcutaneous
oil vehicle injection, PMA infusion into the VMH. Arrow
indicates region of VMH that was analyzed.
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Figure 2.
Quantification of OTR binding in the VMH.
A, Optical density levels of OTR binding in the VMH of
ovariectomized rats treated peripherally with estradiol
(E2) or oil vehicle and centrally with inhibitors
(BIM) or activators (PMA) of PKC.
Treatment groups were Veh (subcutaneous oil vehicle
injection, saline vehicle infusion into the VMH; n = 4), E2 [subcutaneous estradiol (2 µg) injection,
saline vehicle infusion into the VMH; n = 7],
E2 + BIM [subcutaneous
estradiol injection, BIM (200 µM) infusion into the
VMH; n = 4], or PMA [subcutaneous
oil vehicle injection, PMA (200 nM) infusion into the
VMH; n = 5]. Brains were collected 8 hr after drug
infusion and steroid treatment. Data were analyzed by one-way ANOVA and
indicate a significant effect of treatment
[F(3,16) = 12.01;
p < 0.001]; post hoc analysis
indicates that a is significantly different from
b (p < 0.01) and
c (p < 0.001).
B, When compared with the same Veh- and E2-treated
groups as above, infusion of forskolin (Forsk) (200 nM; n = 3), an activator of PKA,
or H89 (E2 + H89) (200 µM;
n = 3), an inhibitor of PKA, into the VMH of
ovariectomized females was without effect on OTR binding in the VMH.
All values are ± SEM.
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Central nucleus of the amygdala
Representative autoradiographs of oxytocin binding in the cAmyg
after the various treatments are pictured in Figure
3. Quantification of binding showed that
infusion of H89, which inhibits PKA activity, significantly decreased
OTR binding in this nucleus compared with vehicle-infused
ovariectomized controls (ANOVA; p < 0.01;
n = 4) (Fig.
4A). The cAmyg
expresses dopamine D1 receptors that are positively coupled to
adenylate cyclase and downstream activation of PKA. Infusion of the D1
antagonist SCH23390 significantly decreased OTR binding density in the
cAmyg of ovariectomized females, compared with vehicle-infused controls
(p < 0.01; n = 3-4) (Fig.
4B). Infusion of forskolin, an activator of PKA,
combined with SCH23390 reversed the effects of the D1 antagonist,
resulting in a return to near basal levels of OTR binding that was
significantly greater than the SCH antagonist alone
(p < 0.05; n = 3) (Fig.
4B). Infusion of the PKC inhibitor, BIM (200 µM), was without effect on OTR binding in the
cAmyg of ovariectomized females (vehicle, 0.295 ± 0.07; BIM,
0.261 ± 0.09).
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Figure 3.
Representative autoradiographs illustrating OTR
binding in the cAmyg. Animals were infused directly into the cAmyg, and
brains were collected 8 hr later and assayed for OTR binding by
quantitative receptor autoradiography. Treatments were as follows:
A, saline vehicle infusion into the cAmyg;
B, H89 (PKA inhibitor) infusion into the cAmyg;
C, SCH23390 (dopamine D1 receptor antagonist) infusion
into the cAmyg; or D, SCH23390 + forskolin infusion into
the cAmyg. Arrow indicates region of cAmyg that was
analyzed.
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Figure 4.
Quantification of OTR binding in the cAmyg.
Ovariectomized rats were treated as follows: A,
Veh 1 (saline vehicle for H89 infusion into the cAmyg;
n = 7), and H89 (H89 infusion into
cAmyg; n = 5); B, Veh
2 (saline vehicle for SCH23390 infusion into the cAmyg;
n = 2); SCH (SCH23390 infusion into
the cAmyg; n = 3), or SCH + Forsk
(SCH23390 + forskolin infusion into the cAmyg; n = 3). Brains were collected 8 hr after drug infusion. There was a
significant effect of H89 infusion [ANOVA;
F(1,10) = 13.96] and SCH23390 infusion
[F(3,7) = 10.62; p < 0.01] when each was compared with its own controls. Post
hoc analysis, **p < 0.01. All values
are ± SEM. Previous treatment with estradiol was found to have no
influence on the effectiveness of H89 treatment (data not shown).
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Experiment II: Assessment of site-specific behavioral effects of
oxytocin infusion
Female sex behavior
Oxytocin infused into the VMH of females primed with a low dose of
estradiol significantly increased lordosis responding (one-way ANOVA
with repeated measures post hoc Kruskal Wallis;
p < 0.001; n = 9) (Fig.
5A) but had no effect on
lordosis when infused into the cAmyg (p > 0.5;
n = 8) (Fig. 5B). The VMH oxytocin-induced increase in lordosis was blocked by previous treatment with the PKC
inhibitor, BIM (p < 0.001) (Fig.
5A). The ability of VMH-infused oxytocin to enhance lordosis
responding was also evident in animals pretreated with the PKC
activator PMA (p < 0.01), although there was no
difference between animals pretreated with PMA or saline vehicle when
they were given oxytocin (data not shown).
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Figure 5.
Effects of oxytocin infusion into the VMH and
cAmyg on female sex behavior. Animals were primed with low doses of
estradiol for 2 d and then pretested for lordosis responding
before infusion of oxytocin (OT; 1 µg) or saline
(Sal) into the VMH or cAmyg. Post-testing was
performed 10-20 min after infusion. Data are presented as the increase
in lordosis amplitude between the pretest and post-test.
A, For animals with indwelling cannulas in the VMH,
oxytocin infusion significantly increased lordosis responding compared
with animals either infused with saline or pretreated for 2 d with
the PKC inhibitor, BIM. ANOVA with post
hoc KruskalWallis; **p < 0.001;
n = 9 for all groups. These results show that
oxytocin infusion into the VMH increases lordosis behavior and this
behavior is blocked by pretreatment with BIM. B, For
animals with indwelling cannulas in the cAmyg, there was no difference
in lordosis responding between those infused with oxytocin
(n = 5) versus saline [n = 4;
F(1,14) = 0.25; p > 0.5]. All values are ± SEM.
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Open-field test
Oxytocin infusion into the cAmyg significantly increased
open-field activity compared with controls, almost tripling the inside crossings/outside crossings compared with saline-infused animals (Mann-Whitney U test; p < 0.01;
n = 9 for oxytocin-infused; n = 6 for
saline-infused) (Fig.
6A). The D1 antagonist
SCH23390 blocked this effect of oxytocin (p < 0.01; n = 8) (Fig. 6A). In contrast,
oxytocin infusion into VMH had no effect on open-field activity when
compared with saline-infused controls (MannWhitney U test;
p > 0.8) (Fig. 6B). There was no
difference in the total number of crossings between any groups,
indicating no changes in general locomotor activity (VMH oxytocin
infusion = 126.2 ± 49.4; saline = 126.0 ± 25.7;
cAmyg oxytocin infusion = 112.3 ± 11.6; oxytocin + SCH23390 = 123.4 ± 14.1; cAmyg saline = 137.7 ± 19.8).
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Figure 6.
Effects of oxytocin infusion into the VMH and
cAmyg on activity in an open field. Animals were placed into the center
of an open-field testing apparatus within 10-20 min of infusion of
oxytocin (1 µg) or saline. The ratio of inside/outside crossings is a
useful overall indicator of activity in an open field. An increase in
the ratio indicates reduced anxiety-like behavior because it
demonstrates that the animals are more willing to explore the center of
the open field. A, For animals with indwelling cannulas
in the cAmyg and infused with oxytocin (n = 9),
there was a significant increase in the ratio when compared with
saline-infused controls (n = 6). This effect was
blocked by pretreatment with the dopamine D1 receptor antagonist,
SCH23390 (n = 8) (**p < 0.01;
ANOVA). There were no differences in overall activity between any
groups. B, For animals with indwelling cannulas in the
VMH, there was no effect of oxytocin infusion on activity in the open
field.
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Elevated plus maze
Although infusion of oxytocin into the cAmyg appeared to increase
the amount of time spent on the open arm of the plus maze and infusion
of the D1 antagonist SCH23390 decreased the amount of time spent in the
open arms when compared with oxytocin-infused controls (SCH23390 + oxytocin = 53.4 ± 6.6 sec, compared with oxytocin = 63.6 ± 12.0 sec and vehicle = 50.3 ± 11.3 sec), these differences failed to reach statistical significance. There were no
differences in total arm entries when comparing oxytocin and saline
infusion (oxytocin + SCH23390 = 14.4 ± 2.8; oxytocin = 10.9 ± 1.8; saline = 11.5 ± 1.8), again indicating no
changes in general locomotor activity as a result of oxytocin infusion. Oxytocin infusion into the VMH had no effect on EPM activity when compared with saline-infused controls (data not shown).
The location of the infusion sites based on cresyl violet-stained
sections resulted in the exclusion of one animal for cAmyg because of
improper cannula placement.
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DISCUSSION |
We report here the regionally specific regulation of OTR binding
and its significance in the control of anxiety and sex behaviors. In
the female rat VMH, OTR binding is reduced by up to 200-fold after
ovariectomy and can be restored fully by replacement of estradiol
(Johnson et al., 1989). A component of the estradiol-induced increase
in OTRs involves increased levels of mRNA (Bale and Dorsa, 1995a,b),
but whether this involves a genomic effect of estradiol is unknown. The
mechanisms by which steroids can alter cellular functions have been
expanded recently to include interactions with cytoplasmic signal
transduction pathways (Zhou et al., 1996; Toran-Allerand et al., 1999).
Our data indicate that estradiol increases oxytocin receptor binding in
the VMH via a PKC-dependent mechanism. Inhibition of PKC in the VMH
blocked the ability of estradiol to increase OTR binding, whereas
activation of PKC significantly increased OTR binding in the absence of
estrogen. Therefore, the estradiol-induced increase in OTR binding in
the VMH functions through activation of PKC and not through genomic
actions of estrogen receptor directly at the OTR promoter.
In contrast to results in the VMH, OTR binding in the cAmyg is not
regulated by estradiol (Bale et al., 1995a,b), and here we report that
it is tonically maintained via a PKA-dependent mechanism involving the
dopamine D1 receptor. This is the first evidence to suggest an
interaction between the OTR and dopamine systems in the cAmyg.
Inhibition of PKA in the cAmyg by H89 significantly decreased OTR
binding when compared with controls. OTR binding was also reduced by
antagonism of the dopamine D1 receptor by SCH23390. Tonic dopamine
input into the cAmyg likely activates PKA through D1 receptors. The
maintenance of OTR binding in the cAmyg appears specific to PKA because
activation or inhibition of PKC had no effect at this brain site.
Oxytocin has been referred to as an "affiliative" hormone because
of its intimate involvement in the regulation of behaviors involving
attachment or close interaction between animals, including anxiety and
sex behavior (Insel and Shapiro, 1992; McCarthy and Altemus, 1997).
Consistent with previous reports (Schumacher et al., 1990; Schulze and
Gorzalka, 1991), we found that infusion of oxytocin into the VMH
increased female sex behavior as measured by lordosis responding.
However, when PKC was inhibited, the enhancing effect of oxytocin on
lordosis was blocked. The results of our binding studies suggest that
the effect of PKC inhibition on lordosis may be caused by the
consequent reduction of OTR expression in the VMH, although we cannot
rule out possible interactions with other components necessary for a
lordotic response. We attempted to increase OTR levels by activation of
PKC with a phorbol ester, with the prediction that oxytocin effects on
lordosis would be enhanced. Although we continued to observe a
significant increase in lordosis amplitude and frequency after oxytocin
infusion, there was no statistical difference in oxytocin effectiveness
between females pretreated with a phorbol ester versus saline. This is likely attributable to a maximal level of OTR expression after the
estrogen treatment. Estrogen has pleiotropic effects on lordosis responding that are not limited to an effect via increased OTR binding
(Pfaff et al., 1994). Nonetheless, our data are the first to
demonstrate that oxytocin stimulation of lordosis behavior involves the
activation of PKC to increase OTR transcription. Previous studies have
shown that PKC activation in the VMH can increase female sex behavior
(Kow and Pfaff, 1998) but have not implicated a specific
neurotransmitter. Estradiol may directly activate the
phosphatidylinositol second messenger pathway leading to PKC activation
(Mobbs et al., 1989) or act indirectly by inducing the expression of
PKC isoforms or fos and jun complexes that can then interact with PKC
to stimulate transcription at AP-1 sites (Gaub et al., 1990) as are
found in the OTR promoter (Bale and Dorsa, 1998). It cannot be
overlooked that inhibition of PKC may also affect other processes
within the VMH that may alter lordosis behavior.
In contrast to the VMH, the cAmyg is a component of the limbic
system and is involved in "emotional" responses, including fear and
defensive behaviors (LeDoux, 1998). Oxytocin has been reported to have
anxiolytic (McCarthy et al., 1996) and antidepressant (Arletti and
Bertolini, 1987) actions, as well as facilitating pair-bonding
(Williams et al., 1994; Insel and Hulihan, 1995) and mother-infant
interactions (Pedersen and Prange, 1979; Fahrbach et al., 1984). Any or
all of these may involve oxytocin action on the cAmyg. These behavioral
responses are modulated by estradiol, but are not dependent on
estradiol, as is the case for VMH regulation of female sex behavior.
The exact CNS site(s) at which oxytocin acts to exert an anxiolytic
effect is unknown. The cAmyg is one of the nuclei of the amygdala that
receives convergent information from several other amygdaloid regions.
It has been proposed that the afferents of this nucleus generate
behavioral responses that reflect the sum of neuronal activity produced
by the amygdala complex (Pitkanen et al., 1997). These observations led
us to speculate that adaptive selective pressure has been in favor of a
constitutive expression of OTRs in the cAmyg to allow for rapid responses to stressful or emotional stimuli by reducing anxiety. The
cAmyg receives direct dopaminergic innervation from the midbrain, and
neurons of this nucleus innervate the dopamine neurons of the
substantia nigra (Rouillard and Freeman, 1995). Conditioned stressors
increase the activity of dopamine neurons projecting to several
components of the amygdala, including the central nucleus (Coco et al.,
1992). Our findings suggest that one function of increased dopamine
release to the cAmyg is the maintenance and/or increase of OTR
expression levels. Oxytocin is also released centrally in response to a
wide variety of stressors (Samson et al., 1985; Nishioka et al., 1998).
Therefore, the oxytocin and dopaminergic systems may act in concert to
reduce anxiety in response to social and environmental stressors. We
found that oxytocin infusion into the cAmyg increased open-field
activity almost threefold and that this effect was not present in
animals pretreated with a D1 receptor antagonist. On the basis of our
binding results, it is likely that the level of OTRs had been
sufficiently reduced by pretreatment with the dopamine antagonist such
that the oxytocin infused into the cAmyg could no longer exert an
anxiolytic effect. Anxiety testing using the same treatments did not
reach significance in the elevated plus maze. We believe that the
open-field test is of more ethological relevance here because female
rodents must leave their burrow and enter into an open field to mate.
Oxytocin is strongly implicated in the control of social behaviors,
suggesting that the difference in magnitude of the anxiolytic effect
between the open field and elevated plus maze may be a function of
measuring different types of anxiety and their potential social
context. In contrast to the cAmyg, oxytocin infusion into the VMH had
no effect on anxiety responses. To our knowledge, this is the first report of a site-specific anxiolytic effect of oxytocin.
A remaining question is the mechanism by which heterogeneous regulation
of OTR expression is achieved in the cAmyg and VMH. The potential for
an undiscovered second OTR gene in the cAmyg cannot be ruled out, and
there is considerable circumstantial evidence to support this view
(Bale et al., 1995a; Verbalis et al., 1995; Verbalis, 1999). An
alternate hypothesis is based on the presence of three large
TG-dinucleotide repeats in the OTR promoter (Bale and Dorsa,
1997). Submillimolar levels of calcium have been shown to regulate DNA
structure at these TG-dinucleotide repeats in the other genes, causing
an alteration in structure of the promoter and brain region-specific
expression of the protein (Dobi and v Agoston, 1998). The heterologous
regulation of OTR expression in the cAmyg and VMH may be a function of
differences in availability of response elements in the promoter caused
by induction of secondary structure.
In summary, we have identified brain region-specific regulation of the
OTR in the VMH and cAmyg and have demonstrated that this heterologous
regulation is critical for two different behavioral effects of
oxytocin. Although the estrogen-dependent OTR expression in the VMH is
induced by activation of the PKC pathway, the receptors in the cAmyg
are dependent on PKA and are tonically activated through dopamine D1
receptors in this nucleus. In the cAmyg, oxytocin acts to decrease
anxiety, but in the VMH it acts to stimulate female sex behavior. A
schematic representation of these results is depicted in Figure
7. Sex receptivity in the female rat is dependent on appropriate levels and duration of exposure to estradiol, thereby temporally restricting its expression to coincide with the
event of ovulation. In contrast, behavioral responses to
anxiety-provoking stimuli such as novelty require a continuously primed
neural substrate to allow for rapid adaptation. OTRs in the VMH and
cAmyg appear to regulate these different behaviors, respectively, and
the distinct signal transduction pathways regulating receptor
expression and binding in each brain region may mediate in part the
ability of oxytocin to exert these differential effects.
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Figure 7.
Summary diagram of effects observed on oxytocin
receptor binding by estradiol and modulators of PKC and PKA and the
resulting effects on behavior. Estradiol increases oxytocin receptor
binding selectively in the VMN but not in the cAmyg. Estrogen action in
the VMN involves PKC and results in increased female sex behavior as
demonstrated by increased lordosis. Dopamine activation of the D1
receptor increases activation of PKA, resulting in increased or
tonically maintained levels of oxytocin receptor binding in the cAmyg.
Oxytocin acting in the cAmyg decreases anxiety. The use of distinct
signal transduction pathways to regulate oxytocin receptor levels in
the VMN and cAmyg may have evolved to allow for constitutive expression
of oxytocin receptors in the cAmyg and an immediate response to
possible life-threatening stimuli, as opposed to the hormonally
constrained control of oxytocin receptors in the VMN that regulate
female reproductive behavior.
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FOOTNOTES |
Received Oct. 13, 2000; revised Jan. 2, 2001; accepted Jan. 23, 2001.
This work was supported by United States Public Health Service (USPHS)
Grant NS-20311 to D.M.D., Molecular and Cellular Biology Training Grant
T32-GM-07270 to T.L.B., and USPHS Grant MH52716 to M.M.M. We thank Cong
Xu for her excellent technical support.
Correspondence should be addressed to Dr. Tracy L. Bale, The Clayton
Foundation for Peptide Biology, The Salk Institute, 10010 N. Torrey
Pines Road, La Jolla, CA 92037. E-mail:
bale{at}salk.edu.
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TOP
ABSTRACT
INTRODUCTION
