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, or 
Opiate Receptors1
Department of Biology, Neuroscience and Behavior Program, University of Massachusetts, Amherst, Massachusetts 01003
Address all correspondence and requests for reprints to: Dr. Sandra L. Petersen, Department of Biology, Neuroscience and Behavior Program, University of Massachusetts, Amherst, Massachusetts 01003.
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Abstract |
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, and/or 
opiate receptors. For these studies, we examined both
intact (n = 3) and ovariectomized, steroid-treated rats. Ten of
the ovariectomized rats were implanted 1 week later (day 0) with
SILASTIC brand (Dow Corning) capsules of estradiol. On the morning of
day 2, half of the estradiol-treated rats were injected with 5 mg
progesterone. All animals were killed at approximately 1530 h on
day 2. We found that cells expressing µ, , and opiate receptor
mRNAs were in all sections that also contained LHRH neurons. In every
case, LHRH neurons were seen to be surrounded by or in close proximity
to cells containing µ, , or mRNAs. However, regardless of
steroid treatment, we found no neurons containing both LHRH mRNA and
mRNAs encoding any of the three receptor subtypes. These results
support the hypothesis that LHRH neurons are regulated indirectly by
opiatergic neurons. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of numerous studies indicate that opiates are inhibitory to LH secretion. For example, it has been shown that the opiate receptor antagonist, naloxone, blocks the negative feedback effects of estradiol (E2) (10) and increases LHRH (11) and LH (12, 13) release. Furthermore, the opiate agonist, morphine, blocks ovulation (14) by interfering with the positive feedback effects of ovarian steroid on LH (15) and LHRH release (16). These findings suggest that endogenous opiates inhibit LHRH neurons and that escape from this inhibition is an important component of the estrogen-dependent trigger for LHRH and LH surge release.
Although there is considerable evidence that opiatergic neurons regulate LHRH release, it remains controversial whether this regulation is direct or indirect. A number of studies have shown that an opioid-noradrenergic interaction in the preoptic area (POA) is an important part of the neural circuitry controlling LH release (9, 17). Opiate antagonists stimulate LH release and concomitantly increase noradrenergic activity (18), whereas morphine blocks both estrogen-induced increases in noradrenergic activity and the LH surge (18, 19). Other studies suggest that a decrease in the release of excitatory amino acids may also mediate the inhibitory effects of opiates on LH release (20). Thus, there is convincing evidence that opiates affect LH release indirectly by inhibiting the release of stimulatory neurotransmitters.
However, there are also data suggesting that LHRH neurons are regulated
directly by opiatergic neurons. The results of several neuroanatomical
studies indicate that ß-endorphinergic neurons synapse on LHRH
neurons in rats (21, 22) as well as in juvenile monkeys (23).
Furthermore, GT1–1 cells, hypothalamically derived transformed tumor
cells with many characteristics of LHRH neurons (24), appear to have
binding sites for receptor-specific ligands (25). Thus, the
neurocircuitry through which opiates modulate LHRH release remains
uncertain.
To resolve the question of whether LHRH neurons are regulated directly
or indirectly by opiate neurons, it is important to determine what
opiate receptor subtype mediates the effects of opiates on LHRH and LH
release. The µ opiate receptor appears to be the major subtype
involved in regulating steroid-dependent LH release (26, 27). However,
µ, , and receptor antagonists can all increase LH release in
ovariectomized (OVX), estrogen-treated rats (27). Likewise, agonists
for each of these opiate receptor subtypes suppress the secretion of LH
in OVX rats (28, 29). Thus, it is presently unclear whether one or
several opiate receptor subtypes mediate the effects of ovarian
steroids on LHRH and LH secretion.
We reasoned that if LHRH neurons are regulated directly by any of the
endogenous opiates, they must synthesize receptors for the µ, , or
opiate receptor. Therefore, in the present studies, we used dual
label in situ hybridization histochemistry (ISHH) to
determine whether LHRH messenger RNA (mRNA) and mRNA encoding µ, ,
and opiate receptors are colocalized in neurons of the POA. We
chose dual label ISHH for these studies rather than dual label
immunocytochemistry primarily because it is easier to verify
colocalization of LHRH and receptor mRNAs than the corresponding
proteins. The mRNA encoding LHRH and various receptors generally
resides in cell bodies or dendrites, whereas receptor proteins are
commonly found in the axon terminal regions of complex neuropil (30),
where it is difficult to determine colocalization with certainty. For
these studies, we examined animals that were OVX, OVX and treated with
E2, or OVX and treated with E2 and progesterone
to control for the possibility that the steroid milieu alters the
expression of opiate receptor mRNAs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For study 1, animals (n = 3) were intact. For studies 2A and B, animals were OVX (n = 15) under metofane anesthesia (methoxyfluor, Pitman Moore, Mundelein, IL) and given steroid replacement as described previously (31, 32). One week after ovariectomy (day 0), some animals (n = 10) were sc implanted with SILASTIC brand capsules (Dow Corning, Midland, MI; id, 1.57 mm; od, 3.8 mm; length, 30 mm) containing E2 (150 µg/ml sesame oil). On day 2, half of the E2-treated animals (n = 5) were given a sc injection of progesterone in oil (5 mg/rat) at 0900 h. All animals were killed at 1530 h. Brains were rapidly removed, frozen on dry ice, wrapped in parafilm, and stored in tightly capped conical tubes at -80 C until cryosectioned.
Study 1
For this study, randomly cycling female rats (n = 3) were
killed at 0900 h. Frozen coronal sections (12 µm) were collected
from the region of the diagonal band of Broca through the anterior
hypothalamus (A 7890-A 6670) (33). Sections were thaw-mounted to
gelatin-coated microscope slides and stored at -80 C until they were
used for hybridization. Adjacent sections were hybridized with
radiolabeled complementary RNA (cRNA) probes specific for µ, , or
opiate receptor mRNAs and digoxigenin-labeled cRNA probes for LHRH
mRNA. For this study, radiolabeled probes were prepared by in
vitro transcription in the presence of 90 pmol
[35S]UTP and 30 pmol UTP as described below. Tissue
preparation, hybridization, and detection methods are also described
below.
Study 2
In study 1, we observed that µ, , and receptor mRNAs
were present in highest abundance in the region containing the organum
vasculosum of the lamina terminalis (OVLT) and the rostral POA (rPOA; A
7470-A 6790) (33). In addition, this region contained most of the LHRH
neurons. Therefore, in studies 2A and B, we obtained 12-µm
cryosections from the rPOA/OVLT region and stored them as described for
study 1. For study 2A, sets of three adjacent sections from OVX and OVX
steroid-treated rats described above were simultaneously hybridized to
digoxigenin-labeled cRNA probes for LHRH mRNA and to
35S-labeled cRNA probes for µ, , and opiate
receptors. However, in this study probes were labeled to a higher
specific activity than those used in study 1. These probes were
prepared by in vitro transcription in the presence of 120
pmol [35S]UTP and no unlabeled UTP. For study 2B, a
second set of adjacent sections from the OVX and OVX steroid-treated
rats was hybridized to probes prepared with 120 pmol
[33P]UTP and no unlabeled UTP.
Tissue preparation
On the day of hybridization, sections were warmed to room
temperature for 10 min, fixed in 4% PBS-formalin for 15 min, treated
with 0.25% acetic anhydride in 0.1 M triethanolamine-0.9%
sodium chloride (pH 8.0), dehydrated and delipidated in a series of
ethanol and chloroform washes, and rehydrated with 95% ethanol. Tissue
sections were then air-dried and hybridized to probes prepared as
described below.
Probe preparation
Radiolabeled cRNA probes for µ, , and opiate receptor
mRNAs. A 450-bp BamHI-Xba fragment
corresponding to the 3'-untranslated region of the µ opiate receptor
mRNA was linearized using ApaI (34). A 773-bp fragment
(corresponding to nucleotides 1351–2124 of the opiate receptor
mRNA that encode 45 bp of the terminal coding region and the
3'-untranslated region) was linearized with HindIII (35, 36). A 983-bp HindIII-SstI fragment
(corresponding to nucleotides 304-1287 of the opiate receptor mRNA
that encode the first extracellular loop to transmembrane domain VII)
was linearized using PstI (36, 37). The specificity of the
µ and opiate receptor cRNA probes was previously verified by
Northern blot analysis (37, 38). The opiate receptor clone was used
previously to map the distribution of opiate receptor mRNA with
in situ hybridization in the rat central nervous system
(36).
Radiolabeled cRNA probes for the opiate receptor mRNAs were prepared by
drying down either 90 (study 1) or 120 (study 2A) pmol
[35S]UTP (DuPont-New England Nuclear, Boston, MA) or 120
pmol [33P]UTP (study 2B; DuPont-New England Nuclear) in a
DNA Speed-Vac (Savant, Farmingdale, NY). One microgram of linearized
template; 5 x transcription buffer; 100 mM
dithiothreitol (DTT); 20 U RNAsin; 0.5 mM ATP, CTP, and
GTP; either 30 pmol (study 1) or no (studies 2A and 2B) UTP; and 10 U
of the appropriate RNA polymerase were added. The µ opiate receptor
complementary DNA (cDNA) was transcribed with SP6 polymerase, the
opiate receptor cDNA with T3 polymerase, and the opiate receptor
cDNA with T7 polymerase. The transcription mixture was incubated for 30
min at 37 C, then a second aliquot of RNA polymerase (10 U) was added
and incubated again for 30 min at 37 C. The template was degraded with
2 U deoxyribonuclease in the presence of 20 U RNAsin, 1 M
Tris-HCl, 1 M MgCl2, and 0.5 µl transfer RNA
(25 µg/µl). The radiolabeled cRNA probes were extracted with
phenol-chloroform, then precipitated twice with NaCl and ethanol and
resuspended in 100 µl 1 mM EDTA and 10 mM
Tris.
Digoxigenin-labeled LHRH cRNA probe. A 330-bp BamHI-HindIII cDNA fragment that corresponds to exons I–IV of LHRH mRNA (39) was linearized with HindIII. Digoxigenin-UTP-labeled cRNA probes were transcribed using 1 µg linearized LHRH cDNA template, T7 RNA polymerase (20 U; Promega, Madison, WI), 5 x transcription buffer, 0.5 mM ATP, CTP, and GTP, 0.1 mM UTP, 0.5 mM digoxigenin-UTP (Boehringer Mannheim, Indianapolis, IN), 100 mM DTT, and 20 U RNAsin. This mixture was incubated at 37 C for 1 h, then an additional 20 U polymerase were added, and the mixture was incubated for another hour at 37 C. Deoxyribonuclease (2 U), in the presence of 20 U RNAsin, was then used to degrade the DNA template. The probe was precipitated with NaCl and ethanol, and resuspended in a solution of 100 µl 1 mM EDTA and 10 mM Tris. The specificity of this probe for LHRH mRNA has been previously verified by Northern blot analysis (40).
Hybridization and probe detection
For these studies, dual labeled ISHH was performed as described
previously (41). For all studies, each section was hybridized with 25
µl hybridization buffer containing 500,000 cpm/section of the desired
radiolabeled opiate receptor cRNA probe and 1 µl digoxigenin-labeled
LHRH probe. The concentration of radiolabeled probe was based on
preliminary studies of the signal to background ratio for various
concentrations of 35S- and 33P-labeled probes.
For subsequent hybridizations, we chose the concentration above which
there was no further increase in signal. The hybridization buffer
contained 2 x SSC (standard saline citrate); 50% (vol/vol)
formamide; 10% dextran sulfate (wt/vol); 250 µg/ml transfer RNA; 500
µg/ml sheared, single stranded, salmon sperm DNA; 1 x
Denhardt’s solution (0.002% Ficoll, 0.002% polyvinylpyrrolidone, and
0.02% BSA); and 50 mM DTT. Sections were hybridized
overnight at 55 C under glass coverslips. After hybridization,
coverslips were removed in 1 x SSC at room temperature. Sections
were washed for 5 min in 1 x SSC at room temperature, incubated
for 5 min, then 20 min in 2 x SSC-50% formamide at 52 C,
followed by two 1-min washes in 2 x SSC at room temperature.
Sections were incubated in ribonuclease buffer consisting of 100
µg/ml ribonuclease A (Boehringer Mannheim), 5 M NaCl, 1
M Tris, and 0.5 M EDTA; washed three times for
5 min each time in 2 x SSC at room temperature; incubated for 5
min in 2 x SSC-50% formamide at 52 C; and washed twice for 5 min
each time in 2 x SSC. After the last wash, sections were
processed for immunocytochemical detection of the digoxigenin-labeled
probes. In studies 1 and 2A, sections were blocked with 2% BSA
(Boehringer Mannheim) in buffer A (100 mM Tris and 150
mM NaCl) with 0.3% Triton X-100 (Sigma Chemical Co., St.
Louis, MO) for 1 h. The sections were rinsed twice for 5 min each
time in buffer A, then incubated in antidigoxigenin-peroxidase
(antidig-POD; 1:200 in 2% BSA in buffer A) for 48 h at 4 C. After
incubation, the sections were rinsed twice for 5 min each time in
buffer A. They were then incubated for approximately 20 min in
3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma). The DAB solution
was prepared by mixing 10 mg DAB in 50 ml 0.1 M Tris (pH
7.6), filtering the solution through Whatman paper (no. 1, Clifton,
NJ), and adding 8 µl hydrogen peroxide. The sections were rinsed
twice in 0.1 M Tris (pH 7.6) for 5 min each time, dipped
quickly in deionized distilled water, and then rinsed for 3 min in 70%
ethanol.
In study 2B, we changed blocking agents because preliminary studies showed that 5% blocking reagent (Boehringer Mannheim) yielded lower nonspecific binding than 2% BSA in buffer A. Therefore, tissue sections in study 2B were blocked with 5% blocking reagent in 0.1 M sodium maleate (pH 7.5) with 0.3% Triton X-100 for 1 h at room temperature. The sections were next rinsed twice in 0.1 M sodium maleate for 5 min each time at room temperature, then incubated for 48 h at 4 C in antidig-POD diluted 1:200 in 2% blocking reagent in 0.1 M sodium maleate. After incubation, the sections were rinsed twice for 5 min each time in 0.1 M sodium maleate. They were then incubated for 4 h in DAB solution as described above. Finally, sections were rinsed twice in 0.1 M Tris, pH 7.6, for 5 min each time, dipped quickly in deionized distilled water, and rinsed for 3 min in 70% ethanol.
After the digoxigenin-labeled probes were detected immunocytochemically, the radiolabeled probes were visualized autoradiographically by dipping the slides in Kodak NTB3 (Eastman Kodak, Rochester, NY) diluted 1:1 with deionized distilled water. Slides were exposed for 2–3 weeks at 4 C, then developed in Kodak Dektol, and fixed in Kodak fixer. Slides were examined microscopically using a x40 objective to determine the number of LHRH neurons that were and were not dual labeled.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Likewise, in studies 2A and B, dual label ISHH of
sections from OVX steroid-treated rats showed no evidence of
colocalization in 785 LHRH neurons, even though probes used for these
studies were of higher specific activity (transcribed in the presence
of 100% either [35S]UTP or [33P]UTP) than
in study 1. Replacement of BSA with blocking reagent in study 2B
reduced the background signal of the digoxigenin-labeled probes, but
did not affect the results of the dual label ISHH. The results of these
studies are summarized in Table 1.
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opiate receptor mRNAs were each present in highest
abundance in the region containing the rPOA/OVLT, specifically in the
region lateral to and above the optic recess and OVLT (nucleus
preopticus, pars suprachiasmatica; A 7470) (33). Cells expressing
receptor mRNAs were also present in this region, but were not as
numerous as those expressing µ or opiate receptor mRNAs. Based on
the findings of study 1, we focused on the rPOA/OVLT in study 2.
Although we saw no evidence of colocalization, we observed that LHRH
neurons were always surrounded by or in close proximity to cells that
expressed µ, , or receptor mRNA. Figure 1 shows
representative sections of the dual label ISHH studies for LHRH mRNA
and µ, , or receptor mRNA in the nucleus preopticus, pars
suprachiasmatica.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, or
opiate receptor mRNA in LHRH neurons despite the abundance of each of
these mRNAs in cells surrounding LHRH neurons. Neither OVX nor steroid
treatment affected colocalization of LHRH and opiate receptor mRNAs.
Thus, it seems likely that opiates regulate LHRH neurons indirectly
through other steroid-sensitive neuronal systems.
Our findings suggest that LHRH neurons are not directly regulated by
opiates. These results contrast with those of previous studies showing
that ACTH- or ß-endorphin-immunoreactive terminals form synaptic
contacts with LHRH neurons in female rats (21), male rats (22), and
juvenile monkeys (23). Furthermore, Lagrange et al. (42)
showed that in guinea pig brain, the selective µ-agonist, DAMGO,
hyperpolarizes hypothalamic LHRH neurons in the presence of
tetrodotoxin. These results suggest that LHRH neurons are regulated by
ß-endorphinergic neurons through a µ opiate receptor. However,
recent studies of LHRH-producing GT1–1 cells indicate that these cells
do not have µ opiate-binding sites (25), nor is LHRH release from
these cells affected by µ opiate receptor agonists (43). Rather,
pharmacological studies suggest that LHRH release from GT1 cells is
regulated by opiates through a receptor (25, 43). Our findings do
not reconcile the results of the studies described above, but instead,
indicate that LHRH neurons in the female rat brain do not appear to
synthesize mRNA for either µ or receptors.
It is possible that the discrepancy between our data and data
suggesting that LHRH neurons are directly regulated by opiates may be
attributable to the sensitivity of the ISHH method. We addressed this
issue by using cRNA probes labeled to high specific activity by
transcribing the cDNA templates in the presence of
[33P]UTP with no competing UTP. Although these probes
readily detected the mRNAs for µ, , and opiate receptors in
cells around LHRH neurons, we cannot rule out the possibility that
levels of these mRNAs are extremely low in LHRH neurons and below the
limits of detectability of our assay. Furthermore, it is possible that
these receptor mRNAs are expressed only under a specific endocrine
milieu not replicated by our steroid treatments, or that they are
expressed for a only a short period of time during the estrous
cycle.
Previous studies on the distribution of opiate receptor mRNAs did not
examine all regions that contain LHRH neruons. However, similar to our
findings, Delfs et al. (38), Mansour et al. (36),
and Minami et al. (44) reported moderate to intense
expression of µ opiate receptor mRNA in the medial POA (mPOA). Also
consistent with our findings, receptor mRNA was observed previously
in the mPOA (36, 45, 46). Receptor mRNA was observed in lower
abundance than and µ and was seen in only scattered cells of the
mPOA (36). Our studies extend previous work by showing that the highest
abundance of µ, , and opiate receptor mRNAs are in the
rPOA/OVLT, a region that includes the anteroventral periventricular
nucleus (PeAV) and the median preoptic nucleus.
Although the possibility that LHRH neurons express very low levels of
opiate receptor mRNAs cannot be ruled out, our findings are more
consistent with the idea that opiates regulate LHRH neurons indirectly,
possibly through the opiate receptor-containing cells residing in the
rPOA/OVLT region. For example, although the PeAV and median
preoptic nucleus are regions of the rPOA/OVLT region that do not
contain LHRH neurons, they are important for estrogen-dependent LH
surge release. The PeAV has a high concentration of
estrogen receptor-containing (47) and estrogen receptor mRNA-containing
neurons (48). Interestingly, we found previously that antiestrogen
implants into this region block estrogen-dependent LH surge release (1, 49) and that estrogen induces the greatest increase in LHRH mRNA before
the LH surge in neurons of this region (2). Furthermore, electrolytic
lesions of structures in the periventricular region of the POA block
the preovulatory LH surge and ovulation (50, 51). Finally, the
PEAV contains sexually dimorphic populations of opiatergic
neurons that are regulated differently in males and females (52). Thus,
it seems possible that the estrogen signal for ovulation is
communicated at least in part to LHRH neurons by the rPOA/OVLT cells,
in which we observed high levels of µ, , and/or opiate
receptor mRNAs.
Although we do not currently know the identity of the opiate receptor mRNA-expressing cells in the rPOA/OVLT region, it seems likely that they are stimulatory neurons. In support of this idea, there is evidence that the inhibitory effects of ß-endorphin may result from a decrease in the release of excitatory amino acids (20). This possibility is particularly interesting in view of recent findings that a subpopulation of LHRH neurons (53) as well as GT1 cell lines (54, 55) express glutamate receptor subtype mRNAs. Thus, it is possible that at least some of the opiate receptor-containing cells in the rPOA are glutaminergic and that opiatergic neurons regulate LHRH neurons indirectly by interfering with the release of this stimulatory neurotransmitter.
It is also possible that the effects of opiates on LHRH and LH release are indirectly mediated by other stimulatory neurotransmitters whose cell bodies reside outside the rPOA/OVLT, possibly noradrenergic and adrenergic neurons. This idea is supported by findings suggesting that opiates act presynaptically to decrease the release of catecholamines. For example, the stimulatory effects of the opiate receptor antagonist, naloxone, on LH release are prevented by interference with the synthesis or binding of epinephrine and norephinephrine (56, 57, 58). Moreover, the magnitude of LH release induced by naloxone depends upon the activity of noradrenergic neurons at the time of treatment (19). Naloxone also increases hypothalamic catecholamine turnover rates (18) and concurrently increases catecholamine and LHRH release from perifused hypothalamic fragments (59). In contrast, morphine blocks both the estrogen-dependent afternoon surge in LH release and the increase in norepinephrine turnover rates that normally accompany the surge (60). Finally, androgenization abolishes the positive feedback effects of estrogen on LH release and alters noradrenergic responses to naloxone (61, 62).
In conclusion, neurons of the rPOA/OVLT that express LHRH mRNA do not
express mRNA encoding µ, , or opiate receptor. These results
support the hypothesis that LHRH neurons are indirectly regulated by
endogenous opiates. Further studies will be required to determine the
phenotypic identity of the cells of the rostral POA that do express
opiate receptor mRNA and to identify the neurocircuitry through which
opiates regulate LHRH and LH release.
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Acknowledgments |
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Footnotes |
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Received November 13, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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