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The Journal of Neuroscience, May 15, 1998, 18(10):3967-3976
Estrogen-Induced Alteration of µ-Opioid Receptor
Immunoreactivity in the Medial Preoptic Nucleus and Medial
Amygdala
Clair B.
Eckersell ,
Paul
Popper , and
Paul E
Micevych
Department of Neurobiology, School of Medicine, and the Laboratory
of Neuroendocrinology, Brain Research Institute, University of
California Los Angeles, Los Angeles, California 90095-1763
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ABSTRACT |
The µ-opioid receptor (µ-OR), like most G-protein-coupled
receptors, is rapidly internalized after agonist binding. Although opioid peptides induce internalization in vivo, there
are no studies that demonstrate µ-OR internalization in response to
natural stimuli. In this study, we used laser-scanning microscopy to
demonstrate that estrogen treatment induces the translocation of µ-OR
immunoreactivity (µ-ORi) from the membrane to an internal location in
steroid-sensitive cell groups of the limbic system and hypothalamus.
Estrogen-induced internalization was prevented by the opioid antagonist
naltrexone, suggesting that translocation was largely dependent on
release of endogenous agonists. Estrogen treatment also altered the
pattern of µ-ORi at the bright-field light microscopic level. In the
absence of stimulation, the majority of immunoreactivity is diffuse,
with few definable µ-OR+ cell bodies or processes. After stimulation, the density of distinct processes filled with µ-ORi was significantly increased. We interpreted the increase in the number of µ-OR+ processes as indicating increased levels of internalization. Using this
increase in the density of µ-OR+ fibers, we showed that treatment of
ovariectomized rats with estradiol benzoate induced a rapid and
reversible increase in the number of fibers. Significant
internalization was noted within 30 min and lasted for >24 hr after
estrogen treatment in the medial preoptic nucleus, the principal part
of the bed nucleus, and the posterodorsal medial amygdala. Naltrexone
prevented the increase of µ-OR+ processes. These data imply that
estrogen treatment stimulates the release of endogenous opioids that
activate µ-OR in the limbic system and hypothalamus providing a
"neurochemical signature" of steroid activation of these
circuits.
Key words:
receptor internalization; neurochemistry of reproduction; steroid hormones; opioid peptides; G-protein-coupled receptors; hypothalamus
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INTRODUCTION |
Neurons in the limbic system and
hypothalamus form a circuit that regulates reproductive behavior. The
processing of sensory and somatic information by these
limbic-hypothalamic neurons is dependent on the hormonal state of the
animal. Circulating steroids regulate the synthesis of several
neurotransmitters in the neurons of the posterodorsal part of the
medial amygdala (MeApd), the bed nucleus of the stria terminalis (BST),
and the medial preoptic nucleus (MPN). In females, estrogen alters the
activity of this circuit resulting in a stimulation of reproductive
behavior. Although a large body of evidence indicates that estrogen
induces the expression of a number of transmitters, including the
endogenous opioid peptides (Dupont et al., 1980 ; Priest et al., 1995;
Micevych et al., 1996), there is a paucity of information about the
time course of estrogen-induced release of these compounds. Determining
the release of neurotransmitters in specific hypothalamic and limbic
nuclei is problematic because the cell groups are compact and the
amounts of neuropeptides released are small (Sinchak et al., 1997b).
Thus, the anatomy of the hypothalamus and the nature of peptidergic
transmission generally preclude the direct measurement of neuropeptide
release from morphologically discrete regions.
Endogenous opioid peptides are widely distributed throughout the limbic
and hypothalamic cell groups and are some of the most extensively
studied transmitters involved in the regulation of female reproduction.
Several lines of evidence implicate endogenous opioid peptides in the
regulation of puberty (Wilkinson and Bhanot, 1982; Bhanot and
Wilkinson, 1983; Sirinathsinghji et al., 1985; Rodriguez et al., 1993),
secretion of luteinizing hormone (Cicero et al., 1979; Kalra and Kalra,
1984; Piva et al., 1985; Zhen and Gallo, 1992) through alterations of
luteinizing hormone-releasing hormone (LHRH) release (Bicknell, 1985;
Kalra, 1986; Piva et al., 1986; Jacobson and Kalra, 1989), and female
sexual behavior (Wiesner and Moss, 1986a,b; Pfaus and Gorzalka, 1987;
Vathy et al., 1991; Pfaus and Pfaff, 1992; Allen et al., 1993; Olster,
1994; Torii et al., 1995, 1996; Sinchak et al., 1997a). Endogenous
opioid peptides also modulate the estrogen-induced expression and
release of other transmitters that alter reproductive behavior, such as cholecystokinin and substance P (Eckersell and Micevych, 1997; Sinchak
et al., 1997b), norepinephrine (Vathy et al., 1991), and serotonin
(Allen et al., 1993).
The major opioid receptor mediating the action of endogenous opioids on
reproduction is the µ-OR. The µ-OR is a typical seven-membrane pass, G-protein-coupled receptor that undergoes agonist-induced receptor internalization. Internalization has been suggested to contribute to receptor desensitization and ligand degradation (Senogles
et al., 1990; Kobilka, 1992; Caron and Lefkowitz, 1993; Lefkowitz et
al., 1993; von Zastrow et al., 1993; Garland et al., 1996). Sternini et
al. (1996) have shown that injections of the nonselective opioid
agonist etorphine produce internalization of µ-ORi in enteric
neurons. We have shown that systemic etorphine injection produces a
redistribution of µ-ORi in the hypothalamus such that the density of
immunoreactive processes was increased. In the medial preoptic area,
treatment of ovariectomized females with exogenous estrogen
qualitatively induced the same type of redistribution of µ-ORi
(Micevych et al., 1997). This redistribution of immunoreactivity
appears to represent the internalization of µ-OR after activation.
Mantyh et al. (1995a,b) and Allen et al. (1997) used the phenomenon of
agonist-induced internalization of substance P receptor
immunoreactivity to determine the activation of neurons after specific
somatosensory stimuli. These studies suggested that monitoring the
redistribution of G-protein-coupled receptors can be used to determine
the neurochemical signature of a stimulus (Allen et al., 1997).
In the present study, we correlated µ-OR internalization with the
increase in the number of discrete µ-OR+ processes, established its
sensitivity to antagonists, and used the redistribution of µ-ORi to
monitor the time course of ligand-activated internalization as a marker
for endogenous opioid release after estrogen stimulation of the limbic
system and hypothalamus.
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MATERIALS AND METHODS |
µ-OR immunohistochemistry
Animals. Adult female Long-Evans rats (65 to 75 d old; Harlan Sprague Dawley, Indianapolis, IN) were ovariectomized
(OVX) by the supplier and shipped to UCLA. On arrival, animals were maintained in the UCLA vivarium under a 12 hr light/dark photoperiod (lights on at 6:00 A.M.) with rodent chow and water available ad
libitum. All of the procedures were approved by the Chancellor's Animal Research Committee at UCLA.
Estrogen stimulation. OVX rats were implanted with a
subcutaneous cannula (polyethylene tubing; Becton Dickinson,
Cockeysville, MD; inner diameter, 0.86 mm; outer diameter, 1.52 mm)
under light ether anesthesia 3 d before estradiol benzoate (EB)
injection. Animals were handled daily, and EB (50 µg in 0.2 ml of
safflower oil) was injected via the cannula. This procedure has been
shown to reduce injection stress and its effects on the enkephalinergic circuit (Eckersell et al., 1996). Immediately after EB injection or
0.5, 1, 2, 4, 6, 12, 24, 48, 72, or 96 hr later (n = 4 per time point) rats were deeply anesthetized (40 mg/kg sodium
pentobarbital) and transcardially perfused with physiological saline
(4°C) followed by 4% paraformaldehyde in 0.1 M
Sörensen's phosphate buffer (4°C). As a control, animals were
injected with the opiate antagonist naltrexone (0.5 mg/ml in saline for
total of 10 mg/kg of body weight) 24 and 4 hr before perfusion and
injected with EB (50 µg in 0.2 ml safflower oil) 4 hr before
perfusion. Brains were removed, post-fixed for 4 hr in the
paraformaldehyde solution, and cryoprotected in 15% sucrose in 0.1 M PBS, pH 7.5. Serial coronal sections (30 µm) through
the medial preoptic area (MPO), and MeApd were obtained on a freezing
microtome (Zeiss Microm, Thornwood, NY) and immunocytochemically
stained.
Immunocytochemical localization of µ-OR. Brain sections
were processed for µ-OR immunocytochemistry using an
affinity-purified antibody raised in rabbit against a synthetic
fragment (LENLEAETAPLP) corresponding to the intracellular C terminus
of rat µ-OR (µ-OR387-398; a gift from C. Evans and B. Anton, UCLA). Free-floating tissue sections were washed in PBS followed
by incubation in 10% normal goat serum, 5% bovine serum albumin, and
0.3% Triton X-100 in PBS with for 30 min. Sections were then incubated
for 48 hr with the affinity-purified µ-OR antibody
(1:200-1:3200) at 4°C. Antiserum was diluted in PBS with 10% normal
goat serum, 5% bovine serum albumin, and 0.3% Triton X-100. After 48 hr, sections were washed in PBS, and alternate sections were processed
with an avidin-biotin-peroxidase complex and either a fluorescent
label for laser scanning confocal microscopy or the chromogen
3,3'-diaminobenzidine tetrahydrochloride (DAB) for light
microscopy.
Sections processed for fluorescence were incubated in blocking buffer
(Tyramide signal amplification kit; NEN Life Science Products, Boston,
MA) and then in biotin-conjugated goat anti-rabbit IgG (Vector
Laboratories, Burlingame, CA; 1:200) for 1 hr. Tissue was then washed
in TBS and incubated in streptavidin-horseradish peroxidase (NEN;
1:100) for 30 min, washed, and then incubated for 8 min in
fluorescein-conjugated tyramide (Tyramide signal amplification kit,
NEN; 1:50). Sections were again washed in 0.1 M Tris buffer
and mounted on Superfrost Plus slides (Fisher Scientific, Houston, TX).
Mounted sections were air-dried and coverslipped using Vectashield
mounting medium (Vector).
Sections processed for bright-field microscopy were incubated with
biotinylated goat anti-rabbit IgG (1:200, 1 hr) followed by an
avidin-biotin complex coupled to horseradish peroxidase (1:50, 1 hr;
Vectastain Elite, Vector) and visualized with the chromogen DAB (Sigma,
St. Louis, MO). Sections were mounted on Superfrost Plus slides (Fisher
Scientific), processed through a series of graded alcohols and xylene,
and coverslipped with Permount mounting medium (Fisher Scientific). To
minimize variability, animals were processed in groups of one animal
from each time point.
Analysis. Specimens were examined with a Zeiss LSM 410 laser- scanning confocal microscope system (Zeiss, Thornwood, NY). The
excitation source was a krypton-argon laser (Coherent, Santa Clara,
CA) with output at 488, 568, and 633 nm. Fluorescein fluorescence was
imaged with a 488 nm emission filter and a 515-540 nm bandpass filter.
The resulting images were created by projecting several optical
sections obtained at different 1 µm intervals through the section in
the z-axis (Fig. 1). To
demonstrate the location of µ-ORi, a single 0.5 µm optical section
in the z-plane was obtained along the major axis and rotated
90° to visualize the interior of the process (Fig.
2). Images were adjusted for brightness
and contrast using the Zeiss LSM-PC program before printing with a Kodak XLS 8600 PF color printer (Eastman Kodak, Rochester, NY). µ-ORi
was considered internalized when the majority of immunoreactivity was
observed in the vesicles within the cytoplasm of neuronal cell bodies
and processes (Fig. 1). An increased number of distinctive µ-OR-immunoreactive somata and processes present in tissue sections stained with DAB were correlated with an increase in the degree of
receptor internalization as shown by fluorescence laser-scanning microscopy. To obtain an estimate of the relative level of
internalization, the density of µ-OR-immunoreactive processes was
determined by superimposing a set of perpendicular lines onto images of
the MPN (Fig. 3) and MeApd at four to six
levels throughout the rostrocaudal extent of the nucleus and counting
all of the distinct, µ-OR-immunolabeled processes that intersect or
touch the line (modified from Eckersell et al., 1992; Priest et al.,
1995). Density was calculated by normalizing the number of processes to
a 100 µm line length. The density of µ-ORi fibers was also
determined in sections throughout the globus pallidus, an area that is
outside of the estrogen-sensitive limbic-hypothalamic circuit.
Treatment groups for each nucleus were compared using one-way ANOVA
with Bonferroni's post hoc comparisons (Sigma Stat, Jandel
Scientific, San Rafael, CA), and differences at the p < 0.05 level were considered significant.
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Figure 1.
Confocal image of cells in the medial preoptic
nucleus stained with antibodies against µ-OR. A, From
an OVX rat, the µ-ORi is associated with the plasma membrane.
B, From an estrogen-treated rat, the µ-ORi is
translocated to the interior of the cell. Magnification, 800×.
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Figure 2.
Confocal photomicrographs of µ-ORi processes in
the medial part of the medial preoptic nucleus from an OVX
(A), OVX plus EB (B), and
OVX plus EB rat pretreated with naltrexone (C).
Immunoreactive fibers were optically sectioned in the
z-axis along the line indicated at the
bottom of each photograph and rotated 90°. The
resulting image (top of each panel) illustrates that the
µ-ORi is associated with the plasma membrane
(arrowheads) in ovariectomized rats
(A). Treatment with estrogen induced a
translocation of the µ-ORi into the interior of the neural process
(B). The neural process had also become more
varicose (B, arrowheads). Neural processes from rats
treated with naltrexone and then estrogen had µ-ORi associated with
the plasma membrane (C, arrowheads). The immunolabeling
in naltrexone-treated rats was consistently less intense than in tissue
from OVX and estrogen-treated rats. Magnification, 2400×.
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Figure 3.
Schematic representation of the quantification
method of µ-OR+ fibers in the medial part of the medial preoptic
nucleus (MPNm). µ-OR+ fibers that touched or
intersected the perpendicular lines superimposed on the µ-ORi plexus
within MPN were counted. The density of immunoreactive fibers was
calculated by dividing the number of processes by the total length of
the lines within µ-ORi plexus. The same type of analysis was applied
to the MeApd. Results are presented in Figure 7. ac,
Anterior commissure; BST, bed nucleus of the stria
terminalis; MPNl, lateral portion of the MPN;
LPO, lateral preoptic area; PS,
parastrial nucleus.
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µ-OR autoradiography
Animals. OVX rats (n = 4 per group)
were injected with EB (50 µg in 0.2 ml of safflower oil). The animals
were decapitated at the time of injections or 6, 12, and 24 hr later.
Brains were removed and blocked in the coronal plane, with the aid of a
Brain Matrix (Activational Systems, Warren MI) into a block extending caudally from the organum vasculosum, lamina terminalis to the mammillary bodies. The brain was then embedded in OCT embedding medium
(Sakura Finetek, Torrance, CA) and frozen on dry ice snow. The tissues
were sectioned at 10 µm with a cryostat, mounted on Superfrost Plus
slides (Fisher Scientific), and were stored at  70°C until used for
receptor binding.
[3H][D-Ala2-N-Me-Phe4,Gly-ol5]-enkephalin
binding. The protocol for µ-OR autoradiography was adapted from
that of Mansour et al. (1995). Slide-mounted tissue sections were
brought to room temperature and incubated for 30 min at room
temperature in 50 mM Tris-HCl, pH 7.5. Sections were
incubated for 60 min at room temperature in TrisHCl containing 20 nM
[3H][D-Ala2-N-Me-Phe4,Gly-ol5]-enkephalin
(DAMGO; Multiple Peptide Systems, San Diego, CA). After incubation,
sections were washed in four changes, 30 sec each, of ice-cold
Tris-HCl. After the final wash, the slides were dipped in two changes
of ice-cold distilled water for 5 sec each to wash off salts. The
slides were dried under a stream of cold air at 4°C and desiccated
overnight at room temperature. Dried slides and 3H
autoradiographic standards (American Radiolabeled Chemicals, St. Louis,
MO) were placed in apposition to Amersham (Arlington Heights, IL)
Hyperfilm for 6 weeks. Films were developed in Kodak D-19 developer and
fixed with Kodak fixer. Nonspecific binding was determined by
processing paired serial sections as described above, except that 20 µM nonradioactive DAMGO was added to the binding
solution.
Analysis. Optical densities were measured on sections
through the MPO and MeApd taken at four different anteroposterior
levels using a computer-assisted image analysis system and expressed as
nanocuries per milligram of standard. Sections used for autoradiography were thionin-stained and used to define the MPO and MeA. Specific binding in each section was calculated by subtracting the nonspecific binding values obtained from its paired section from the total binding
values. The average specific binding for each MPO or MeA was
calculated, and the specific binding values across treatment groups
were compared using one-way ANOVA (Sigma Stat; Jandel Scientific, San
Rafael, CA).
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RESULTS |
Antibody specificity
No positive structures were observed in sections processed for
immunocytochemistry without the µ-OR antiserum. Preabsorption of the
µ-OR antiserum with the synthetic peptide fragment used to generate
antibodies prevented immunostaining in tissue specimens and has
previously been reported (Sternini et al., 1996; Olive et al., 1997).
Moreover, the appearance and distribution of µ-ORi in this study is
similar to the distribution Mansour et al. (1995) observed using a
different polyclonal antibody generated against 63 amino acids
(µ-OR335-398) of the C terminus of the cloned µ-OR.
Distribution of µ-ORi in the limbic-hypothalamic circuit
µ-OR immunocytochemistry revealed a plexus of distinct
immunoreactive fibers in the MPN, principal nucleus of the BST (BSTp), and MeApd (Fig. 4). The MPN µ-ORi
plexus extended the full rostrocaudal length of the medial preoptic
area but was more pronounced in the dorsal aspect of the medial part of
the MPN (MPNm). The medial preoptic plexus began rostrally in fibers
surrounding the anteroventral periventricular nucleus and ended
caudally at the anterior hypothalamic area. No µ-ORi was observed in
the cell-dense, central portion of the MPN (MPNc) although the rest of
the MPNm contained a large amount of immunoreactivity (Fig. 4). The
µ-ORi plexus extended dorsally and laterally from the posterior MPN
into the striohypothalamic nucleus and continued into the BSTp. µ-ORi
cell bodies were sparsely distributed in the MPN. The greatest density
of immunoreactive cell bodies was observed in a continuous band from
the most caudal portion of the MPN through the striohypothalamic
nucleus.
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Figure 4.
Photomicrographs of the medial preoptic area
stained with antibodies against the µ-OR. A,
µ-OR-immunoreactive fibers are concentrated in the medial part of the
medial preoptic nucleus, surrounding the central part
(MPNc), which is devoid of labeled fibers. µ-ORi
fibers form a dense plexus dorsolateral to the MPNc. B,
Other concentrations of µ-ORi fibers at the level of the MPNc,
including the striohypothalamic nucleus (StHy) and the
principal portion of the bed nucleus of the stria terminalis
(BST). f, Fornix;
LV, lateral ventricle.
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µ-ORi fibers form a plexus in the principal and the lateral strial
portion of the BST (Fig. 4). The stria terminalis, a major neuronal
pathway containing reciprocal projections between the amygdala and the
BST, also exhibited dense µ-ORi staining. A plexus of µ-ORi fibers
was also observed in the dorsolateral aspect of the MeApd. µ-ORi was
also present in the medial, central, and posteromedial cortical nuclei
of the amygdala.
The globus pallidus, which is outside of the estrogen-sensitive,
limbic-hypothalamic circuit and has a dense network of µ-ORi fibers,
was used as a control for the effects of estrogen on the distribution
of µ-ORi in these studies.
Cellular distribution of µ-ORi in ovariectomized rats
Confocal microscopy confirmed earlier work that the cellular
localization of µ-ORi in unstimulated animals was primarily
associated with cell membranes of neural processes and cell bodies
(Sternini et al., 1996). In untreated OVX females, the preponderance of µ-ORi appeared as patchy areas of fluorescence associated with the
plasma membrane of cell bodies and presumptive dendrites throughout the
MPN, BST, and MeApd (Figs. 1, 2). Scattered among the
membrane-associated µ-ORi profiles were thin varicose fibers in which
µ-ORi filled the cell body or process and that did not appear to be
associated with the membrane.
Bright-field microscopy revealed a µ-ORi that was generally diffuse
and only occasionally associated with distinct neuronal processes and
cell bodies in the nuclei of the limbic-hypothalamic circuit (Figs.
5, 6).
µ-ORi labeling within the OVX animals could be divided into two
types: (1) the majority of labeling was on fibers with smooth, diffuse
outlines and weakly labeled cells; and (2) the minority of labeling was
in distinct immunoreactive processes with intensely labeled varicose
fibers.
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Figure 5.
Bright-field photomicrographs of µ-OR
immunocytochemical localization in the medial MPN, dorsal to the
central part of the MPN in the area quantified (Fig. 3). OVX animals
(A) had a low density of distinct µ-OR+ fibers
(A, arrows). Although a number of distinct µ-OR+
fibers were detected (arrows), the majority of µ-OR
immunoreactivity was diffuse (A, C, D, arrowheads).
Treatment with estrogen produced a dramatic increase in the density of
varicose µ-OR+ fibers at 4 hr (B), but by 72 hr
(C) the distribution resembled tissue from
ovariectomized rats. The estrogen-induced increase was blocked by
pretreatment with naltrexone (D). Scale bar:
D, 25 µm.
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Figure 6.
Bright-field photomicrographs of µ-OR
immunocytochemical localization in the posterodorsal medial amygdala in
OVX (A) and estrogen-treated rats (B, C,
D). Estrogen induces an increase in the number of µ-OR+
fibers 4 hr after estrogen (B); by 72 hr
(C) the density is similar to OVX levels.
Naltrexone blocks the estrogen effect (D) and
induces a significant decrease in the density of fibers. Scale bar:
D, 50 µm.
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Pattern of µ-ORi after stimulation
Etorphine treatment has been shown to induce activation and
internalization of µ-ORi in cells transfected with the µ-OR
in vitro (von Zastrow et al., 1994) and enteric neurons
in vivo (Sternini et al., 1996). In the brain, etorphine
induced a rapid and reversible change in the subcellular localization
but not the tissue distribution of µ-ORi (Micevych et al., 1997;
Keith et al., 1998). After etorphine treatment, the number of cell
bodies and processes in which the µ-ORi was localized within the
structure was dramatically increased. In these structures, the
immunoreactivity was not associated with the plasma membrane but was
localized within processes in a pattern that is consistent with
internalized receptors (Mantyh et al., 1995a,b; Sternini et al., 1996;
Allen et al., 1997; Olive et al., 1997; Keith et al., 1998). This
pattern of receptor internalization was correlated at the light
microscopic level with an increase in the density of distinct varicose
fibers throughout the brain, including the cortex, the globus pallidus,
thalamus, lateral hypothalamus, and central amygdala. Moreover, the
density of the µ-ORi fibers within the plexuses in the MPN, BST, and
MeA was increased.
As with etorphine, the tissue distribution of µ-ORi was not changed
by treatment with estrogen. Moreover, as with etorphine, confocal
microscopic examination revealed that µ-ORi was primarily internalized in cell bodies and processes of the MPN, BSTp, and the
MeApd after estrogen treatment (Figs. 1, 2). At the light microscopic
level, the number of distinctly labeled µ-ORi fibers was increased in
the limbic-hypothalamic nuclei after estrogen treatment (Figs. 5, 6).
In contrast to etorphine-treated rats, the increase in µ-ORi
processes after estrogen treatment was restricted to steroid-responsive
regions of the brain. No increase in varicose µ-ORi fibers was
observed in the cortex, thalamic nuclei, central amygdaloid nuclei, or
globus pallidus (Table 1).
Estrogen stimulation significantly increased the density of µ-ORi
processes in the MPN and MeApd within 30 min (Fig.
7, Table 1). This elevation of µ-ORi
processes remained for 24 hr and then declined to basal levels by 48 hr. The density of µ-ORi processes remained at this low level for the
duration of the 96 hr experiment. Treatment of ovariectomized rats with
naltrexone (10 mg/kg) before stimulation with estrogen not only
prevented the increase of µ-ORi processes within the MPNm and MeApd
but reduced the density of fibers to a significantly lower level than
in unstimulated animals at 0 hr (Fig. 7).
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Figure 7.
Graphic representation of the density of µ-ORi
fibers in the medial part of the MPN (MPNm) (solid line)
and the posterodorsal medial amygdaloid nucleus (MeApd) (dashed
line) after estrogen treatment. The moment of EB injections is
time 0. The density of distinct µ-OR+ fibers was significantly
elevated by 30 min and peaks at 4-6 hr. The density of µ-OR+ fibers
is basal by 48 hr. The bars at the 4 hr time point
illustrate the density of µ-OR+ fibers in the MPNm (solid
bar) and MeApd (open bar) in OVX plus EB rats
pretreated with naltrexone 24 and 4 hr before perfusion. The density of
µ-OR+ fibers in the naltrexone-treated rats is significantly lower
than in ovariectomized rats in the MPN and MeApd. Each point is the
mean ± SEM (n = 4 per time point).
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µ-OR-specific binding
Specific binding with [3H]DAMGO was observed
in the MeA and MPO at all time points (Fig.
8). Estrogen injection had no significant overall effect on µ-OR-specific binding levels either in the MeA or
in the MPO at any of the time points investigated.
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Figure 8.
Histogram showing [3H]DAMGO
binding in the MeA (solid bar) and MPO (open
bar) at 0, 6, 12, and 24 hr after subcutaneous injection of 50 µg of EB. No significant change in [3H]DAMGO
binding was observed in either the MeA or MPO after estrogen injection.
Each point is the mean ± SEM (n = 4 per time
point).
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DISCUSSION |
To our knowledge, the present communication is the first report
that indicates the µ-OR is redistributed in response to a naturally
occurring stimulus. Translocation of µ-ORi from the plasma membrane
to the interior of the cell after estrogen treatment was interpreted as
agonist-induced receptor internalization. To date, most studies that
examine opioid receptor internalization have been in transfected cells
in vitro (von Zastrow et al., 1993; Arden et al., 1995;
Keith et al., 1996, 1998). Such studies indicate that after the initial
agonist-receptor interaction and signal transduction, µ-ORs are
typically internalized through the process of endocytosis into the
endosomal compartment. The ligands are dissociated from the receptors,
and some of the receptors are recycled back to the plasma membrane.
There is a paucity of studies that have examined the activation of
opioid receptors in vivo (Sternini et al., 1996; Keith et
al., 1998).
The present study used the idea, proposed by Mantyh et al. (1995b) and
Allen et al. (1997), that translocation of G-protein-coupled receptors
is a pharmacologically specific index of neuronal activity. Furthermore, agonist-induced endocytosis of a signal-transducing receptor is a valid method for identifying the anatomical components of
highly specific neuronal circuits. These authors quantified the number
of endosomes with the confocal microscope before and after stimulation.
In the present study, we used the redistribution of immunoreactivity,
which resulted in an increase of µ-OR+ processes, as a measure of
µ-OR activation in the limbic-hypothalamic circuit in response to
estrogen stimulation. Several lines of evidence suggest that the
increase of µ-ORi fiber density we measured in this study is a marker
for receptor internalization. First, in alternate sections using the
confocal microscope, we observed a translocation of µ-ORi from the
plasma membrane to the intracellular compartment after estrogen
treatment. The intracellular localization of µ-ORi produced a
concentration of immunoreactivity that allowed for the visualization of
a greater number of fibers. Internalization of µ-ORi was therefore
correlated at the light microscopic level with an increase in the
number of varicose µ-ORi fibers after stimulation. Second, the same
morphological reorganization and increase in the number of µ-ORi
processes observed in the limbic-hypothalamic cell groups after
estrogen treatment was seen after etorphine treatment. Third,
naltrexone blocked the estrogen-induced increase in immunoreactive
processes, strongly suggesting that estrogen provoked the release of
endogenous opioids that activated and induced the internalization of
the µ-ORi. Fourth, correlation of ultrastructural and light
microscopic localization of the somatostatin sst2A receptor showed that
diffuse labeling of processes at the light microscopic level is related
to a membrane location of the receptor and distinct labeling of
processes is correlated with internalized receptor immunoreactivity
(Dournaud et al., 1997).
A process, other than internalization, that could produce an increase
in immunoreactivity after estrogen treatment is an increase of µ-OR
expression. It could be argued that newly synthesized receptors would
be localized in vesicles before their insertion into the membrane, and
so the immunoreactivity would be predominately intracellular. However,
estrogen stimulates µ-OR mRNA expression 48 hr after treatment
(Quinones-Jenab et al., 1997) (protein levels were not reported), and
by the 48 hr time point in the present study, the estrogen effect had
passed (Fig. 7). Moreover, [3H]DAMGO binding
levels do not change for 24 hr after estrogen treatment (Fig. 8). The
rapidity of the estrogen effect (30 min), similar time course and
pattern of µ-ORi redistribution after etorphine, and sensitivity to
naltrexone also argue against the possibility of increased µ-OR
expression as the cause of the increased number of immunoreactive
fibers.
In contrast to the extended time course of internalization after
estrogenic activation, Mantyh et al. (1995b) showed that within 60 min
after somatosensory stimulation, substance P receptor was no longer
internalized. The extended internalization of the µ-OR may be
attributable to the prolonged clearance time of the steroid. A 50 µg
injection of EB maintains significantly elevated serum estrogen levels
for >24 hr, which return to baseline by 48 hr after estrogen treatment
(Priest et al., 1995), suggesting that estrogen may induce continuous
release of endogenous opioid peptides and µ-OR internalization
throughout this period.
Previous studies have implicated estrogen and progesterone in the
regulation of opioid receptors in the MPN. Estrogen decreases and
progesterone further decreases the number of µ-OR binding sites in
the MPN (Hammer, 1984, 1990; Weiland and Wise, 1990; Piva et al., 1995;
but see Shen et al., 1995). These studies demonstrate a downregulation
of the µ-OR. In contrast, the present study examined short-term
alterations of the cellular distribution of µ-OR induced by estrogen.
Agonist-induced internalization removes receptors from the plasma
membrane, preventing further stimulation by ligands, but the numbers of
receptors are not significantly altered (Lefkowitz et al., 1993; von
Zastrow and Kobilka, 1994). Several studies have reported that estrogen
does not alter the number of µ-OR in the short term (Mateo et al.,
1992; Shen et al., 1995). Similarly, our results that
[3H]DAMGO binding in the MPN and MeA did not
fluctuate during the 24 hr after estrogen treatment support the idea
presented here that early estrogen regulation of the µ-OR is the
result of receptor internalization and desensitization and not
downregulation.
The results of the present study suggest that estrogen induces a rapid
release of the endogenous opioid peptides in the MPN, BST, and MeA that
activate µ-OR in these regions. An important question is which
endogenous opioids are being stimulated by estrogen. Endorphins,
enkephalins, and dynorphins all have an N terminus (YGGFM/L) that binds
with moderate specificity to the µ-OR,  -OR, and  -OR. There are
several potential candidates in the limbic-hypothalamic circuit.
Estrogen increases the expression of the enkephalin precursor preproenkephalin mRNA in the MPNm, MeApd, and ventromedial hypothalamus (Romano et al., 1989; Eckersell et al., 1994; Priest et al., 1995; Holland et al., 1997). Furthermore, the immunocytochemical distribution of  -endorphin increases on estrus, the day after estrogen levels peak during the estrous cycle (Ge et al., 1993), and -endorphin fiber density in the MPNm varies with sex steroid levels (Hammer and
Cheung, 1995). These fibers originate from cells in the arcuate nucleus
that also respond to estrogen stimulation (Hammer et al., 1991).
Because of their selectivity, endogenous peptides have been assigned to
different opioid receptors; enkephalins are considered the endogenous
ligands for -OR. -Endorphin binds to µ-OR and -OR with
similar affinity (Lee and Smith, 1980; Patterson et al., 1983; Smith et
al., 1983). Another likely candidate is the novel endogenous opioid
peptide endomorphin-1, the putative endogenous ligand for the µ-OR
that is concentrated in the hypothalamus (Zadina et al., 1997). The
characterization of the endogenous ligand(s) that are released in the
limbic-hypothalamic circuit to activate µ-OR will require further
investigation.
Both direct and indirect evidence suggests that endogenous opioids
regulate sexual receptivity through activation of µ-OR. Systemic
treatment of estrogen-primed OVX rats with naloxone, an opioid
antagonist, facilitated sexual receptivity, as measured by lordosis
behavior (Wiesner and Moss, 1986a), and site-specific injections of
-FNA, a µ-OR antagonist, into the MPN also increased lordosis
behavior in estrogen-primed female rats (Hammer et al., 1989). Further
injections of the selective, endogenous µ-OR agonist endomorphin-1
into the MPN blocked lordosis in estrogen- and progesterone-primed females (K. Sinchak, C. Evans, N. Maidment, and P. Micevych,
unpublished observations). Also, infusions of -FNA into the MPN
attenuated the estrogen-stimulated release of cholecystokinin, a
neuropeptide that facilitates lordosis in the MPN (Sinchak et al.,
1997b). These findings, therefore, are consistent with the hypothesis that endogenous opioid peptides released in the MPN act at the µ-OR
to decrease sexual receptivity.
It is clear that a number of transmitters in addition to the endogenous
opioids are involved in the integrated signaling of the
limbic-hypothalamic nuclei that regulate reproductive behavior. The
present results suggest that by studying the redistribution of
receptors in this circuit, it may be possible to define the neurochemical signature (Allen et al., 1997) and temporal sequence of
hormonal activation of the brain. Such an analysis will allow for a
more complete understanding of estrogen activation of neural circuits.
| |
FOOTNOTES |
Received Dec. 9, 1997; revised Feb. 27, 1998; accepted March 5, 1998.
This research was supported by National Institutes of Health Grants
NS21220 and HD07228 and National Institute on Drug Abuse Grant DA05010.
We thank Wesley Tsai, Paul Nguyen, Victor Yu, and Allison Norell for
their technical assistance, and Drs. Kevin Sinchak and Christopher
Evans for their insightful comments on this manuscript. The University
of California Los Angeles Mental Retardation Research Center Media Core
was helpful with the preparation of illustrations.
Correspondence should be addressed to Dr. Paul E Micevych, Department
of Neurobiology, University of California Los Angeles School of
Medicine, Los Angeles, CA 90095-1763.
| |
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Top
Abstract
Introduction
