Vol. 92, Issue 2, 493-503, February 2002
Sexually dimorphic effects of morphine and MK-801: sex
steroid-dependent and -independent mechanisms
Deborah N.
D'Souza1,
Richard E.
Harlan1,3, and
Meredith M.
Garcia2,3
Departments of 1 Structural and Cellular Biology and
2 Otolaryngology, and 3 Neuroscience Program, Tulane
University School of Medicine, New Orleans, Louisiana 70112
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ABSTRACT |
Rats show gender
differences in responses to morphine and the
N-methyl-D-aspartate receptor antagonist
dizocilpine (MK-801); the role of sex steroids in mediating these
differences is unclear. We tested the overall hypothesis that
circulating gonadal steroids determine the gender differences in
morphine- and MK-801-induced behavior and c-Fos expression. Morphine
caused a greater expression of c-Fos in the striatum of intact males
than of that females, which was independent of sex steroids. MK-801
completely inhibited morphine-induced c-Fos in intact females but only
caused partial inhibition in intact males; castrated males showed
complete inhibition, which was reversed by testosterone, but gonadal
steroids had no effect on this response in females. In thalamus, there
was a large sex difference in the response to MK-801 that was
independent of gonadal steroids. Behavioral responses to morphine were
greater in males, but responses to MK-801 were greater in females; both were sex steroid independent. These findings show significant sex
differences in response to morphine and MK-801 that are mediated by sex
steroid-dependent and -independent mechanisms, which may be important
in treatment outcomes of drug addiction.
immediate-early genes; c-Fos; striatum; thalamus; opiate-mediated
behavior
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INTRODUCTION |
MORPHINE IS A COMMON
PALLIATIVE for severe pain and is also a major drug of abuse. Use
of morphine for pain relief is limited, however, because of development
of tolerance and the risk of respiratory depression. Previous studies
have documented sex differences in the response to morphine in rats,
mice, monkeys, and humans. Gender differences in morphine-induced
antinociception have been reported, with males generally displaying
greater effects than females (5, 10, 13, 33, 34, 37, 41, 49,
53). Sex differences have also been reported in morphine
tolerance and dependence (18), in the discriminative
stimulus effects of morphine (17), in morphine-induced
conditioned place preference (15), in self-administration of opiates (1, 45), and in morphine-induced hypothermia
(35), although in a recent study by Stewart and Rodaros
(57) no gender differences were observed in the stimulant
effects of morphine. Sex differences in the respiratory effects of
morphine in humans have also been reported (20), although
in a recent retrospective study by Zacny (64), no gender
differences were reported in miosis or rate of respiration after
intravenous administration of morphine. In this same study, however,
the subjective effects of morphine were reported to be greater in
female subjects than in males. Sex differences in patterns of drug
abuse, including abuse of opiates, also have been reported, with
approximately twice as many men as women using illicit drugs (6,
39, 51, 56). The role of steroid hormones in these gender
differences is controversial and may be species specific and dependent
on the effect studied (3, 16, 31, 33, 48, 49). Further information on sex differences and the roles of gonadal steroids in the
responses to morphine may provide more tailored approaches to pain
relief and to treatment of drug abuse.
Previous studies have demonstrated that some of the effects of morphine
can be attenuated by antagonists of
N-methyl-D-aspartate (NMDA) glutamate receptors
(e.g., 4, 22, 41, 42). Gender differences have also been reported in
the effects of the noncompetitive NMDA receptor antagonist dizocilpine
maleate (MK-801). Honack and Loscher (30) reported greater
behavioral effects in females, who showed hyperlocomotion, head
weaving, and ataxia after an injection of MK-801 (0.1 mg/kg ip), as
opposed to males, who showed almost no behavioral effects in response
to a similar dose of the drug. However, in deer mice, the inhibitory
effects of MK-801 on analgesic responses are greater in males than in
females (41). These data thus suggest that some of the
MK-801-induced effects are sensitive to gender. This issue is important
because of the wide use of MK-801 in studies on NMDA receptors.
Earlier studies from this and other laboratories have shown that
morphine induces expression of immediate-early genes (IEG), especially
c-Fos, in the rat caudate putamen (CPu) and thalamus (4, 11, 22,
25, 42). MK-801 and the competitive NMDA receptor antagonist
NPC-17742 attenuate morphine-induced c-Fos expression in the rat
forebrain (5, 22, 42), and our laboratory has shown that
this attenuation is sexually dimorphic (22). In the
present study, we tested several hypotheses. The overall hypothesis is
that circulating gonadal steroids determine the gender differences in
the behavioral and c-Fos responses to morphine, MK-801, and their
combination. Thus castrated rats of both sexes were compared, to test
the hypothesis that some gender differences are independent of
circulating gonadal steroids. In addition, rats were either left
gonadally intact or castrated and replaced with the gender-appropriate
gonadal steroid, to test the hypothesis that some sex differences
depend on circulating steroids. This approach also allowed us to test
the hypothesis that responses to morphine, MK-801, or their combination
are modified by gonadal steroids, in either gender.
Induction of c-Fos expression in the midline-intralaminar thalamic
nuclei and the striatum suggests that morphine activates thalamostriatal circuits. Indeed, direct evidence for this hypothesis has recently been presented (26). Although the role of
this circuit in the behavioral or physiological responses to morphine has not been determined, it is possible that this circuit is involved in the rewarding properties of morphine. It has been reported recently
that microinfusion of an antisense oligonucleotide to c-fos
mRNA into the nucleus accumbens, part of the ventral striatum, reduced
the rewarding effects of morphine, as determined by conditioned place
preference (59). Thus a focus on potential sex differences in this circuit may shed light on the neural mechanisms of gender differences in patterns of substance abuse in humans.
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METHODS |
Treatment of animals.
Adult male or random-cycling female Sprague-Dawley rats were housed in
group cages in the vivarium of the Tulane University Medical School,
with controlled temperature and light-dark cycles of 12:12 h (lights on
0600; lights off 1800). Animals were provided with unlimited access to
food (Purina rat chow) and tap water. All animal studies were approved
by the Tulane Medical Center Advisory Committee for Animal Resources
and conformed to the National Institutes of Health Guide for the
Care and Use of Laboratory Animals. All efforts were made to
minimize animal suffering and to reduce the number of animals used.
Male rats were either left intact or anesthetized with methoxyflurane
and castrated by removal of the testes and testicular fat with a single
2-cm midscrotal incision. Half the castrated rats were given daily
injections of 2 mg/kg testosterone propionate dissolved in sesame oil
for a total of 8 days. Female rats were either left intact or
anesthetized with methoxyflurane, ovariectomized via bilateral dorsal
incisions, and implanted subcutaneously with either empty Silastic
capsules or capsules containing crystalline estradiol (5 mm releasing
length, 0.24 mm wall thickness). Eight days after surgery, the animals
were assigned to one of four treatment groups (4 animals/group) and
injected according to the following protocol: 1) vehicle
(water, ip) followed by vehicle (water, sc) (V-V); 2)
vehicle followed by morphine sulfate (10 mg/kg sc) (V-M); 3)
MK-801 (0.2 mg/kg, ip) followed by vehicle; or 4) MK-801
followed by morphine. The second injections were administered 30 min
after the first injections. Morphine sulfate was obtained from
the National Institute on Drug Abuse, and MK-801 was obtained from
RBI/Sigma Chemical. Steroids were obtained from Sigma Chemical.
Behavioral analysis.
Behavioral observations were made immediately after the first injection
and extended until the time of perfusion (i.e., 2 h after the
second injection). The intensity of Straub tail was classified as
"not present," "weak," or "strong." The sedative effects of
morphine were classified as "typical" (rats isolated from others in
the cage and generally unreactive to other rats), "intense" (rats
immobile but somewhat reactive to handling), and "catatonic"
(immobile and unreactive to handling, with respiratory depression).
General locomotor activity and motor coordination were also noted.
Results were analyzed statistically with the 
2 test.
Tissue preparation.
Two hours after the second injection, the rats were deeply anesthetized
with an overdose of pentobarbital sodium (100 mg/kg ip; Nembutal,
Abbott). The rats were then perfused with 0.025 M PBS, pH 7.2, for 3 min, followed by 3% buffered paraformaldehyde for 6 min. Brains were
dissected out, blocked at the midpontine level, postfixed in
paraformaldehyde for 2 h at room temperature, cryoprotected in
30% sucrose at 4°C until they sank, frozen with crushed dry ice, and
stored at 
70°C until they were sectioned.
Immunocytochemistry.
The brains were cut on a freezing microtome (60-µm coronal sections)
and processed for immunocytochemistry as described previously (25). Sections were washed in PBS and incubated for 1 h at
room temperature in blocking serum [normal goat serum, 15 µl/ml in 0.4% Triton (TX)-PBS]. The sections were then incubated in rabbit anti-c-Fos (sc-52, Santa Cruz; 1:10,000 dilution) at 4°C for 2 days.
After being washed in PBS, the sections were incubated for 1 h with the
secondary antibody (biotinylated goat anti-rabbit IgG, 1:400 in 0.4%
TX-PBS; Vector), washed in PBS again, and incubated in ABC solution
(1:100 in 0.4% TX-PBS; Vectastain Elite, Vector) for 1 h. The sections
were finally washed in Tris buffer 0.05 M, pH 7.6, and then immersed in
0.05% 3,3'-diaminobenzidine tetrahydrochloride and 0.005%
H2O2 to visualize the reaction product. The
reaction was stopped with PBS, and the sections were mounted from 10 mM sodium acetate containing 0.1% Dreft's detergent onto chrom-alum subbed slides. The sections were dried using a slide warmer, dehydrated in 100% ethanol for 2 min, and cleared in Histoclear (National Diagnostics) for 10 min before coverslipping with Permount (Fisher).
Data analysis.
The immunocytochemical data were examined with a Nikon Optiphot
microscope. The regions of the brain examined for immediate-early gene
(IEG) expression were the dorsomedial CPu and the midline intralaminar
nuclei of the thalamus. The numbers of labeled cells per unit area were
counted using the NIH Image program according to the methods described
previously (9). With the use of image analysis, the
desired regions of interest were outlined on the brain images, i.e.,
within the dorsomedial CPu (height 1.66 mm; width 1.30 mm), the
reuniens nucleus (height 0.58 mm; width 0.84 mm), the rhomboid nucleus
(height 0.51 mm; width 0.51 mm), the central medial nucleus (height
0.35 mm; width 1.30 mm), and the paraventricular nucleus (height 0.53 mm; width 0.66 mm), and counts were taken within the circumscribed
region. The data were analyzed statistically with one-way or three-way
ANOVA. For the treatment groups in which ANOVA showed significance
(which was all of the groups), ANOVA was followed by post hoc
analysis with the Fisher's paired least-significant difference,
Scheffé's, or Bonferroni/Dunn tests.
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RESULTS |
IEG expression in the CPu.
In males, morphine caused a significant induction of c-Fos expression
in the dorsomedial CPu of rats that were intact (Figs. 1A and
2A), castrated (Fig.
1A), or castrated and treated with testosterone (Fig.
1A). There was no significant difference across the three
groups in the response to morphine. Thus the hypothesis that gonadal
steroids modify the c-Fos response to morphine in males was not
supported. MK-801 followed by vehicle did not induce c-Fos in any group
(Fig. 1A). MK-801 significantly inhibited morphine-induced c-Fos expression in the dorsomedial CPu in all three groups (Fig. 1A); however, the magnitude of the MK-801-mediated
inhibition was dependent on gonadal hormones. In intact male rats,
MK-801 significantly decreased morphine-induced c-Fos expression,
although the number of c-Fos-positive cells was still greater than in
vehicle-treated rats (Figs. 1A and 2, A and
B). After castration, there was a complete inhibition of
morphine-induced c-Fos expression in males (Figs. 1A and
2C); however, in castrated males that received testosterone replacement, MK-801 showed the same partial inhibition of morphine's effects as was seen in the intact males (Figs. 1A and
2D). Thus the hypothesis that gonadal steroids modify the
interaction between morphine and MK-801 was supported in males.
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Fig. 1.
Mean ± SE number of c-Fos immunoreactive (ir) cells
in the dorsomedial caudate putamen (CPu) of male (A) and
female (B) rats. Data represent the number of c-Fos cells
after treatment with vehicle followed by vehicle (V-V), vehicle
followed by morphine (V-M), dizocilpine maleate (MK-801) plus vehicle
(MK-V), and MK-801 plus morphine (MK-M). Morphine alone significantly
increased c-Fos expression in the dorsomedial CPu of male and female
rats, although the effect was larger in males than in females
(P < 0.05). In male rats, MK-801 significantly
decreased morphine-induced c-Fos expression, although the number of
c-Fos-positive cells was still greater than in vehicle-treated rats.
After castration, MK-801 completely inhibited morphine-induced c-Fos
expression in males. After treatment with MK-801 and morphine, there
was a significant difference between the number of c-Fos-positive cells
in intact males vs. castrated males, but there was no significant
difference between intact males and castrated males treated with
testosterone (Cast + Test). MK-801 completely inhibited
morphine-induced c-Fos expression in females regardless of gonadal
hormone status (Ovx, ovariectomized; Ovx + E, ovariectomized and
treated with estrogen). The number at the bottom of each bar represents
the number of animals in each group. *P < 0.005 vs.
corresponding V-V, **P < 0.005 vs. corresponding V-M,
and +P = 0.05 vs. MK-M in intact
males.
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Fig. 2.
Photomicrographs of the dorsomedial CPu of intact male rats treated
with morphine (A) or MK-801 plus morphine (B),
castrated male rats treated with MK-801 plus morphine (C),
castrated plus testosterone-treated male rats injected with MK-801 plus
morphine (D), and intact (Int) female rats treated with
morphine (E) or MK-801 plus morphine (F).
Treatment with morphine alone induced a larger c-Fos response in intact
males (A) compared with intact females (E) in the
dorsomedial CPu. MK-801 significantly reduced morphine-induced c-Fos
expression in intact males (B) and completely inhibited this
expression in castrated males (C) and in intact females
(F). LV, lateral ventricle.
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In females, morphine caused a significant induction of c-Fos in the
dorsomedial CPu in rats that were intact, ovariectomized, and
ovariectomized with estrogen treatment (Fig. 1B). Thus the hypothesis that gonadal steroids modify the c-Fos response to morphine
in females was not supported. However, the magnitude of the effect of
morphine was smaller in the intact females than in the intact males
(Table 1; Figs. 2, A and
E), and this sex difference was not eliminated by
gonadectomy or with or without hormone replacement. Thus the hypothesis
that the sex difference in the c-Fos response to morphine is determined
by circulating gonadal steroids was not supported. MK-801 completely
inhibited morphine-induced c-Fos response in all three groups of
females (Fig. 1B and 2F). In females, regardless
of gonadal hormone status, MK-801 inhibition of morphine-mediated c-Fos
expression was significantly greater than in intact or castrated plus
testosterone-treated males. However, this sex difference was eliminated
by castration (Table 1). Thus the hypothesis that the sex difference in
the interaction between morphine and MK-801 is mediated by gonadal steroids was supported.
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Table 1.
Summary of treatment conditions in which the c-Fos response in the CPu
was significantly greater in males than in females
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Three-way ANOVA revealed highly significant main effects of sex
(P < 0.001), drug treatment (P < 0.001), and sex-drug interactions (P < 0.01) in c-Fos
expression in the dorsomedial CPu.
IEG expression in the thalamus.
Morphine alone caused a significant induction of c-Fos in the central
medial nucleus in intact and castrated males (Fig.
3A) and in the rhomboid
nucleus of castrated males (Fig.
4A). In female rats, morphine
induced c-Fos expression in the central medial nucleus of
ovariectomized rats (Fig. 3B) and in the rhomboid nucleus of
intact rats (Fig. 4B). To analyze the data further for
potential sex differences, we performed a separate ANOVA on each
thalamic nucleus, collapsing across the hormone-treatment groups. This analysis revealed that morphine significantly induced c-Fos in the
central medial nucleus in both sexes (mean = 22.9 ± 6.5 for V-V in males, 93.9 ± 15.7 for V-M in males; 17.0 ± 2.2 for V-V in females, and 72.8 ± 5.6 for V-M in females).
There was no sex difference in the response to morphine. The effect of
morphine was not blocked by MK-801 in males; however, the analysis is
more complicated in females because MK-801 greatly induced c-Fos
expression in the midline-intralaminar thalamic nuclei (see
below). There was no effect of morphine in the reuniens or
paraventricular nuclei in either sex (Figs.
5 and 6).
Examples of the responses to the different treatments in castrated male
and female rats are shown in Fig. 7.
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Fig. 3.
Mean ± SE number of c-Fos immunoreactive cells in
the central medial nucleus of male (A) and female
(B) rats. Morphine alone induced c-Fos in the central medial
nucleus of ovariectomized females and intact and castrated males.
MK-801 alone induced significant c-Fos expression in all groups of
female rats. Morphine significantly attenuated MK-801-induced c-Fos
expression in females, although this effect was significantly less in
ovariectomized rats than in intact or estrogen-replaced rats. The
number at the bottom of each bar represents the number of animals in
each group. *P < 0.05 vs. corresponding V-V,
**P < 0.005 vs. corresponding V-V,
***P < 0.05 vs. corresponding MK-V, and
+P < 0.05 vs. MK-M in intact female and
MK-M in Ovx+E.
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Fig. 4.
Mean ± SE number of c-Fos immunoreactive cells in
the rhomboid nucleus of male (A) and female (B)
rats. Morphine alone induced c-Fos in the rhomboid nucleus of castrated
males and intact females. MK-801 alone induced a significant increase
in c-Fos expression in female rats and intact and castrated male rats.
Morphine significantly attenuated the MK-801-induced c-Fos expression
in females and intact males. This attenuation was significantly less in
ovariectomized rats than in intact or estrogen-replaced females. The
number at the bottom of each bar represents the number of animals in
each group. *P < 0.05 vs. corresponding V-V,
**P < 0.005 vs. corresponding V-V,
***P < 0.05 vs. corresponding MK-V, and
+P < 0.05 vs. MK-M in intact females and
MK-M in Ovx+E.
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Fig. 5.
Mean ± SE number of c-Fos immunoreactive cells in
the reuniens nucleus of male (A) and female (B)
rats. MK-801 alone induced significant c-Fos expression in male and
female rats. Morphine significantly attenuated MK-801-induced c-Fos
expression in females and castrated males. This attenuation was
significantly less in ovariectomized rats than in intact females or
estrogen-replaced rats. The number at the bottom of each bar represents
the number of animals in each group. **P < 0.05 vs.
corresponding V-V, ***P < 0.01 vs. corresponding MK-V,
and +P < 0.05 vs. MK-M in intact
females.
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Fig. 6.
Mean ± SE number of c-Fos immunoreactive cells in
the paraventricular nucleus of male (A) and female
(B) rats. MK-801 alone induced a significant increase in
c-Fos expression in female rats. Morphine significantly attenuated
MK-801-induced c-Fos expression in females. The number at the bottom of
each bar represents the number of animals in each group.
**P < 0.05 vs. corresponding V-V, ***P < 0.05 vs. corresponding MK-V.
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Fig. 7.
Photomicrographs of the midline intralaminar thalamic
nuclei of castrated male rats treated with vehicle (A),
morphine (B), MK-801 (C), or MK-801 plus morphine
(D) and ovariectomized female rats treated with vehicle
(E), morphine (F), MK-801 (G), or
MK-801 plus morphine (H). Morphine induced c-Fos expression
in the central medial (Cm) and rhomboid (Rh) nuclei but not in the
reuniens nucleus (Re) in castrated males (B) and
ovariectomized females (F). MK-801 alone induced a far
greater amount of c-Fos expression in female rats (G) than
in male rats (C) in all 3 nuclei illustrated. Morphine
significantly attenuated MK-801 induced c-Fos expression in females
(H), especially in the rhomboid nucleus. 3V, 3rd ventricle.
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Significant sex differences were found in the response to MK-801 and to
MK-801 plus morphine in all four thalamic nuclei (Figs. 3-6; Table
2). In females, but not in males, MK-801
greatly induced c-Fos expression in all four nuclei. The greater c-Fos
response to MK-801 in females was independent of circulating gonadal
steroids in all four nuclei (Fig. 7, C and G).
Thus the hypothesis that gender differences in the c-Fos response to
MK-801 are mediated by gonadal steroids was not supported. In all four
nuclei of female rats, morphine significantly decreased the c-Fos
expression induced by MK-801 (Fig. 3-6). However, the extent of
this decrease was modulated by circulating gonadal steroids. In the
reuniens, rhomboid, and central medial nuclei, the number of
Fos-positive cells after MK-801 plus morphine was significantly greater
in the ovariectomized rats than in the intact females or
estradiol-replaced rats (P < 0.05). Moreover, in the
absence of gonadal steroids, the combination of MK-801 plus morphine
induced significantly more Fos-positive cells in these three nuclei in
females than in males (P < 0.01; Table 2). Thus the
hypothesis that gonadal steroids modify the interaction between
morphine and MK-801 was supported in females.
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Table 2.
Summary of treatment conditions in which the c-Fos response in thalamic
nuclei was greater in females than in males
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Behavioral observations.
After morphine administration, the animals became quiet and stuporous.
They lost their righting reflex and displayed Straub tail. We looked at
the presence of Straub tail during the first 60 min and at 120 min
after the injection and categorized this response as "not present,"
"weak," or "strong." After collapsing across steroid treatment
groups, there was a significant sex difference in the intensity of
Straub tail in response to morphine (Table 3). At both time points, there was a
significantly stronger effect on Straub tail in males than in females
(P < 0.05). We did not test for effects of gonadal
steroids.
Administration of MK-801 before morphine significantly enhanced the
sedative response to morphine in both sexes (Table 3). Only 5 of the 12 male rats and none of the female rats given MK-801 plus morphine showed
a "typical" response to morphine, compared with all 24 of the rats
given vehicle-morphine. Moreover, MK-801 plus morphine induced
catatonia in five male and eight female rats, whereas morphine by
itself never induced catatonia in either sex. The frequency of
"intense" sedation plus "catatonia" was significantly greater
in females than in males after MK-801 plus morphine. In addition,
MK-801 greatly increased the intensity and duration of Straub tail
produced by morphine in female rats both during the first 60 min
(2 = 8.0, P < 0.05) and at 120 min
(2 = 16.1, P < 0.01) after
morphine administration. Thus the combination of MK-801 plus morphine
produced a more profound behavioral effect in females than in males. We
did not test for effects of gonadal steroids.
We also saw a significant behavioral response to MK-801 in females, in
which, within 20 min after the MK-801 injection, the rats started to
stumble, weave their heads back and forth, wobble, and show significant
hyperactivity for 2.5 h after the injection, until the time of
perfusion. There were no obvious effects of gonadal hormones on
behaviors induced by MK-801 in females. After the administration
of MK-801 alone in males, we did not see any obvious behavioral
effects. This sex difference persisted in the gonadectomized rats.
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DISCUSSION |
Sex differences in the behavioral and IEG responses to
morphine alone were independent of circulating gonadal hormones. The larger morphine-induced c-Fos expression in the dorsomedial CPu in
males was not modified by gonadal hormones. Morphine also induced a
stronger behavioral effect in males than in females, and this was not
altered by gonadal steroids. These findings are consistent with
previous studies that have reported greater morphine-induced antinociception in males (5, 10, 13, 33, 34, 37, 41, 49,
53), which also has been reported to be independent of gonadal
hormones (3, 33, 49). They are also consistent with
findings that gonadal steroids do not alter sexually dimorphic opioid-induced respiratory effects in women (20), the
potency of morphine in a drug discrimination test in rats
(17), or the initiation or maintenance of opiate
self-administration in rats (58).
The lack of effect of sex steroids on opiate-mediated analgesia,
respiratory depression, and reward is in contrast to their effects on reproduction through opioidergic systems. Both
endogenous opioids and opioid receptors in the hypothalamus are
important in reproductive function in both sexes, and a number of
studies have shown regulation of mu opioid receptor expression by
estrogen in several hypothalamic nuclei; however, estrogen does not
regulate opioid receptor expression in most other regions of the
forebrain, including the striatum (8, 21, 32, 52).
Estrogen has also been shown to have an acute effect on mu opioid
receptor distribution in the medial preoptic nucleus of the
hypothalamus, the bed nucleus of the stria terminalis, and the
posterodorsal medial amygdala (23), and estrogen has been
shown to modulate mu opioid receptor activity in cultured neurons from
the arcuate nucleus, through interactions with the protein kinase A
pathway (62). Thus sex steroids may exert region-specific
activational effects on opioid receptors, but these effects spare the
striatum. Gender differences in the analgesic and reward properties of
opiates thus seem more likely to be due to the organizational effects of sex steroids rather than their activational effects. Steroids are
known to exert organizational effects on the brain, which are mediated
by the presence of sex steroids during certain critical periods of
development. Evidence exists to suggest that this may be an underlying
mechanism for gender differences in some responses to morphine
(40), which may be expressed as sex-specific differences in the number and/or distribution of opioid receptors in the brain (28, 65), although this has not been examined in the
striatum. Another possible mechanism for gender-specific responses to
opiates could be in pharmacokinetics; however, no gender differences
have been shown to exist in morphine pharmacokinetics in the rat
(14, 15).
In contrast to the lack of effect of gonadal steroids on
morphine-mediated c-Fos expression, we saw an androgen-dependent sex
difference in the ability of MK-801 to block morphine-induced c-Fos
expression in the dorsomedial CPu. An involvement of glutamate and NMDA
receptors in morphine-induced IEG expression in the striatum has been
reported (4, 22, 42). The cerebral cortex and the
midline-intralaminar thalamic nuclei are the major sources of
glutamatergic inputs to the striatum. Whereas the cerebral cortex
contains low levels of mu opioid receptors, the medial thalamic nuclei
contain the highest concentration of mu opioid receptors in the brain
(7). The central medial thalamic nucleus projects
selectively to the dorsomedial CPu (2), where systemic morphine administration induces expression of IEGs. Recently, a role
for thalamostriatal projections in morphine-induced striatal c-Fos
expression has been demonstrated by infusion of the mu opioid receptor
antagonist 
-funaltrexamine into selected midline-intralaminar thalamic nuclei (24). These results suggest that
morphine-induced IEG expression in the dorsomedial CPu is subsequent to
activation of thalamostriatal glutamatergic projections. This is
consistent with the finding that infusion of MK-801 into the CPu blocks
IEG induction by systemic morphine (4). It is also
consistent with morphine-induced activation of c-Fos, especially in the
central medial thalamic nucleus (25; present results), and with
increased blood flow to the medial thalamus induced by the mu receptor
agonist hydromorphone in humans (54). The inability of
MK-801 to block morphine-induced c-Fos expression in the central medial
nucleus is consistent with a thalamic effect of morphine independent of NMDA receptors. Our results of sex differences in the striatal c-Fos
response to morphine suggest the possibility of sex differences in
thalamostriatal mechanisms. Sex differences in the abundance of mu
opioid receptors in the human thalamus have been reported (65), with females showing higher levels than males,
although this difference disappeared with age. The existence of sex
differences in mu opioid receptors in the thalamus and striatum of rat
brain has not been investigated, to our knowledge.
Potential mechanisms by which androgens regulate the striatal response
to MK-801 include regulation of NMDA receptors and/or the activity of
thalamostriatal neurons. There is much evidence in the literature
suggesting that estrogens regulate NMDA receptors in the brain (e.g.,
27, 36). It was therefore surprising that we saw no effect of estrogens
on the ability of MK-801 to block morphine-induced IEG expression in
the dorsomedial CPu. Evidence suggests that androgens can regulate
expression of NMDAR1 subunits in the hypothalamus (38) and
MK-801 binding in the hippocampus (50). Whether similar
regulation occurs in the striatum, where androgen receptors are low or
absent (55), is not known. Very low levels of androgen
receptor mRNA-containing cells were reported in the thalamic
paraventricular nucleus and none in the central medial, rhomboid, and
reuniens nuclei of the thalamus (55). However, there are
various neuropeptidergic inputs from the hypothalamus to the
midline-intralaminar thalamic nuclei, including substance P,
somatostatin, neurotensin, and -endorphin. Androgens can regulate -endorphin neurons in the brain (12, 46, 47) and can
increase -endorphin immunoreactivity in the fiber plexus of the
thalamus (29). Thus androgens may exert effects on midline
intralaminar thalamic nuclei through peptidergic inputs. Altered
activity of thalamostriatal neurons might explain a complete block
of the morphine-induced IEG expression by MK-801 in the CPu of
castrated males as opposed to an incomplete block in intact males.
Similarly, estrogen-regulated neuropeptide inputs to the thalamus may
underlie the decreased ability of morphine to block MK-801-induced IEG expression in the midline-intralaminar thalamus in ovariectomized rats
(Figs. 5-7).
In intact female rats, the administration of MK-801 alone produced a
robust increase in c-Fos expression in the cortex (22, 26), and reuniens, rhomboid, central medial, and paraventricular nuclei of the thalamus. This effect was sexually dimorphic and independent of gonadal steroids in females. MK-801 induced no obvious
behavioral responses in males but induced uncoordinated hyperactivity
in females. Although sex differences in MK-801-induced behavior have
been reported earlier (19, 30), this is the first report
that has systematically investigated the effect of gonadal hormones on
IEG expression and behavioral effects in response to MK-801. We found
that the behavioral sex difference in response to MK-801 persists in
gonadectomized rats, with or without appropriate steroid replacement.
Honack and Loscher (30) also reported that these sex
differences are not likely to be dependent on the estrous cycle or on
differences in drug metabolism between males and females. MK-801 has
been found to increase serotonin metabolite levels in brain regions in
both male (63) and female (43) rats. However, we know of no direct comparisons of the serotonin-releasing effects of
MK-801 in male vs. female rats. MK-801-induced IEG expression in the
thalamus in females could be due to an activation of 5-HT (serotonin) receptors in this region of the brain. Moreover,
the behaviors induced by MK-801 are similar to those induced by 5-HT agonists such as 8-OH-DPAT (61), L-tryptophan,
or 5-hydroxytryptophan (60), and antagonists of the 5-HT1A
receptor block the behavioral effects of MK-801 (44).
These reports thus suggest that the behavioral effects of MK-801 may be
modulated by serotonergic systems. Whether serotonergic mechanisms
underlie the sexual dimorphism in the IEG response to MK-801 is not known.
In conclusion, we observed sex differences in MK-801 and
morphine-induced behaviors and IEG expression in the brain, and some of
these gender differences appear to be mediated by gonadal steroids, whereas others are independent of circulating gonadal steroids. These
studies may have therapeutic implications for the treatment of drug
addiction and for pain control by opiates.
| |
ACKNOWLEDGEMENTS |
These studies were supported in part by Louisiana Educational
Quality Support Fund (95) Grants RD-A-29 (to M. M. Garcia) and National Institute on Drug Abuse Grant DA-11939 (to R. E. Harlan).
| |
FOOTNOTES |
Address for reprint requests and other correspondence: R. E. Harlan, Dept. of Structural and Cellular Biology, Tulane Medical School, 1430 Tulane Ave. SL49, New Orleans, LA 70112-2699 (E-mail: harlanre{at}tulane.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/japplphysiol.00565.2001
Received 4 June 2001; accepted in final form 10 October 2001.
| |
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