Oxytocin in the nucleus accumbens shell reverses
CRFR2-evoked passive stress-coping after partner
loss in monogamous male prairie voles
Oliver J. Bosch, University of Regensburg
Joanna Dabrowska, Rosalind Franklin University of Medicine and Science
Meera E. Modi, Harvard University
Zachary V. Johnson, Emory University
Alaine C. Keebaugh, Emory University
Catherine E. Barrett, Emory University
Todd H. Ahern, Emory University
Jidong Guo, Emory University
Valery Grinevich, Heidelberg Univ
Donald Rainnie, Emory University
Only first 10 authors above; see publication for full author list.
Journal Title: Psychoneuroendocrinology
Volume: Volume 64
Publisher: Elsevier | 2016-02-01, Pages 66-78
Type of Work: Article | Post-print: After Peer Review
Publisher DOI: 10.1016/j.psyneuen.2015.11.011
Permanent URL: https://pid.emory.edu/ark:/25593/rwrs0
Final published version: http://dx.doi.org/10.1016/j.psyneuen.2015.11.011
Copyright information:
© 2015 Elsevier Ltd.
This is an Open Access work distributed under the terms of the Creative
Commons Attribution-NonCommercial-NoDerivatives 4.0 International License
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Accessed December 10, 2021 3:02 AM EST
HHS Public Access
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Neuroscience. Author manuscript; available in PMC 2016 December 17.
Published in final edited form as:
Neuroscience. 2015 December 17; 311: 422–429. doi:10.1016/j.neuroscience.2015.10.047.
Regional Differences in Mu and Kappa Opioid Receptor Gprotein Activation in Brain in Male and Female Prairie Voles
Thomas J. Martin1, Tammy Sexton2, Susy A. Kim1, Amie L. Severino1, Christopher M.
Peters1, Larry J. Young3, and Steven R. Childers2
Author Manuscript
Thomas J. Martin: tjmartin@wakehealth.edu; Tammy Sexton: tsexton@wakehealth.edu; Susy A. Kim:
sukim@wakehealth.edu; Amie L. Severino: aseverin@wakehealth.edu; Christopher M. Peters: chrpeter@wakehealth.edu;
Larry J. Young: lyoun3@emory.edu; Steven R. Childers: childers@wakehealth.edu
1Pain
Mechanisms Laboratory, Department of Anesthesiology, Wake Forest University Health
Sciences, Winston-Salem, North Carolina
2Department
of Physiology and Pharmacology, Wake Forest University Health Sciences,
Winston-Salem, North Carolina
3Silvio
O. Conte Center for Oxytocin and Cognition, Center for Translational Neuroscience,
Department of Psychiatry and Behavioral Sciences, Yerkes National Primate Research Center,
Emory University, Atlanta, Georgia
Abstract
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Prairie voles are unusual mammals in that, like humans, they are capable of forming socially
monogamous pair bonds, display biparental care, and engage in alloparental behaviors. Both mu
and kappa opioid receptors are involved in behaviors that either establish and maintain, or result
from pair bond formation in these animals. Mu and kappa opioid receptors both utilize inhibitory
G-proteins as signal transduction mechanisms, however the efficacy by which these receptor
subtypes stimulate G-protein signaling across the prairie vole neuraxis is not known. Utilizing
[35S]GTPγS autoradiography, we characterized the efficacy of G-protein stimulation in coronal
sections throughout male and female prairie vole brain by DAMGO and U50,488H, selective mu
and kappa opioid agonists, respectively. DAMGO stimulation was highest in forebrain, similar to
that found with other rodent species. U-50,488H produced greater stimulation in prairie voles than
is typically seen in mice and rats, particularly in select forebrain areas. DAMGO produced higher
stimulation in the core versus the shell of the nucleus accumbens in females, while the distribution
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Corresponding author: Thomas J. Martin, PhD, tjmartin@wakehealth.edu, Tel: 336-716-8554, FAX: 336-716-6744.
Thomas J. Martin wrote the manuscript and assisted with experimental design and data analysis.
Tammy Sexton performed [35S]GTPγS membrane binding experiments, autoradiography studies, quantified autoradiograms, and
sectioned vole brains.
Susy A. Kim maintained the prairie vole colony and assisted with tissue dissection and perfusion.
Amie L. Severino performed prairie vole perfusions, dissection, Nissl staining and sectioned perfused brains and wrote portions of the
manuscript.
Christopher M. Peters performed prairie vole perfusions, dissection, and Nissl staining and wrote portions of the manuscript.
Larry J. Young edited and wrote portions of the manuscript.
Steven R. Childers designed [35S]GTPγS autoradiography and membrane binding experiments, provided blinding of Ms. Sexton for
image analysis and quantification, provided data analysis, and wrote portions of the manuscript.
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Martin et al.
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of U-50,488H stimulation was the opposite. There were no gender differences for U50,488H
stimulation of G-protein activity across the regions examined, while DAMGO stimulation was
greater in sections from females compared to those from males for nucleus accumbens core,
entopeduncular nucleus, and hippocampus. These data suggest that the kappa opioid system may
be more sensitive to manipulation in prairie voles compared to mice and rats, and that female
prairie voles may be more sensitive to mu agonists in select brain regions than males.
Keywords
guanosine 5′-O[γ-35S] triphosphate; second messenger; signaling; autoradiography; DAMGO;
U50; 488H; rodent; monogamy; social behavior; pair bonding
1
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Prairie voles (Microtus orchogaster) are relatively unusual among rodent species in that the
male and female form lifelong, socially monogamous pair bonds after mating (Young et al.,
2001, McGraw and Young, 2010, Johnson and Young, 2015). Prairie voles also, like
humans, engage in alloparental behavior with both male and female involved in rearing of
the young (Young et al., 2001, Young and Wang, 2004, Ahern and Young, 2009, Ahern et
al., 2011). Due to these human-like social behaviors, the neurobiology of prairie voles
related to formation of pair bonds has been studied in some detail and these studies have
shown that oxytocin, vasopressin, and their receptors have prominent roles in pair bond
formation and maintenance (Young et al., 2001, Lim et al., 2004, Young and Wang, 2004,
Donaldson et al., 2010). Additionally, both mu and kappa opioid receptors are involved in
these processes. Mu opioid receptor activation in limbic forebrain regions is required for
pair bond formation in female prairie voles, while kappa opioid receptor activity is required
for male-male aggression following pair bond formation with a female (Burkett et al., 2011,
Burkett and Young, 2012, Resendez et al., 2012, Resendez et al., 2013). The role of opioid
receptor subtypes in other brain areas in prairie vole behavior and neurobiology is an area of
increased interest and investigation.
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The neuroanatomical distribution of mu and kappa opioid receptor mRNA and protein has
been examined in prairie vole brain, and their distribution is reasonably comparable to that
found in the rat and mouse (Inoue et al., 2013). While opioid receptor density is generally
highest in brain regions that are involved in the prominent pharmacological effects of
opioids in the central nervous system, the efficacy by which opioids stimulate second
messenger signaling through receptor activation does not always correlate with receptor
density. The efficacy by which opioid agonists are able to activate second messenger
systems is often determined using agonist stimulated binding of guanosine 5′-O[γ-35S]
triphosphate ([35S]GTPγS) binding (Sim et al., 1995). This assay has the advantage in that it
can be performed autoradiographically in tissue sections, and thereby agonist efficacy can be
determined throughout the neuraxis for several compounds in the same animal. Such studies
are valuable in that they determine relative agonist efficacies throughout the brain for a
variety of receptor subtypes efficiently, and these experiments have been used extensively to
compare receptor-G-protein coupling efficiency between animals or across pharmacological
treatments (Breivogel et al., 1999, Sim-Selley et al., 2000).
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In this study, we determined the efficacy of the mu opioid agonist DAMGO and the kappa
opioid agonist U50,488H to stimulate G-protein activity using [35S]GTPγS binding in
striatal membranes and using autoradiography in discreet regions throughout the brain in
male and female prairie voles, and confirmed receptor selectivity for each agonist at
appropriate concentrations of opioid antagonists. Additionally, anatomical differences
between males and females were examined for both receptor subtypes.
2. Experimental Procedures
2.1. Subjects
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A prairie vole colony was established at Wake Forest University Health Sciences (WinstonSalem, NC) using animals imported from a colony at Emory University (Atlanta, Georgia)
originally derived from wild caught animals from Illinois. Subjects consisted of adult,
sexually naïve male (N=8) and female (N=8) prairie voles (age 12–18 weeks) from the
Wake Forest University Health Sciences colony weighing 40–60 g at time of sacrifice. All
animals used for this study were housed in same sex groups of 2–4 animals and kept on a
reversed 10:14 hr light:dark cycle (dark 03:00–17:00) in a temperature and humidity
controlled vivarium within an AAALAC approved facility. The male and female pairs were
housed separately but in the same room. Female prairie voles are induced into estrous in the
presence of male urine, but without direct contact with males the ovaries are quiescent and
these animals had not initiated an ovarian cycle. Males were sexually naïve as well. All
animals were given ad libitum access to high fiber rabbit chow pellets (ProLab 5P25,
LabDiet, St. Louis, MO), alfalfa cubes (Country Acres Feed, Brentwood, MO) and water.
Bedding material consisted of ¼ inch corncob pellets (Bed-o’Cobs ¼”, The Andersons Lab
Bedding, Maumee, OH), paper nesting material (Crink-l’Nest, The Andersons Lab Bedding)
and cotton fiber nestlets (Ancare Corp., Bellmore, NY). Each cage also contained one red
polycarbonate tube (3 inch diameter, 6 inch length, Bio-Serv, Flemington, NJ) for nesting
and burrowing. All procedures were approved by the Animal Care and Use Committee of
Wake Forest University (Winston-Salem, NC) and were in accordance with the Guide for
the Care and Use of Laboratory Animals as adopted and promulgated by the National
Institutes of Health (Bethesda, MD).
2.2 Identification and verification of neuroanatomical brain regions
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As there is currently no brain atlas available for the prairie vole, identification and
verification of anatomical regions examined using the [35S]GTPγS autoradiography assay
was accomplished using Nissl staining at 5 coronal levels of prairie vole brain in
conjunction with a standard rat brain stereotaxic atlas (Paxinos and Watson, 1998). For this
purpose four prairie voles were anesthetized with a lethal dose of sodium pentobarbital (100
mg/kg, i.p.). A blunt 23 gauge needle connected to a 20 ml syringe by 2 mm diameter silicon
tubing was inserted into the left ventricle. The tip of the needle was stabilized with a
hemostat and the vole was slowly perfused with 30 mL of 0.1 M phosphate buffered saline
(PBS, pH = 7.4 at 4°C) followed by 30 mL of 4% formaldehyde fixative solution in 0.1M
PBS. Brains were removed and post-fixed for 12 hours and then transferred to 30% sucrose
solution in 0.1M PBS for cryoprotection. Serial 50 μm coronal brain sections were cut on a
cryostat (Leica Microsystems, Buffalo Grove, IL) and thaw-mounted on plus slides for
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histological processing. Nissl stained sections were rehydrated with 0.1M PBS for 10 min,
post-fixed onto slides with 4% formaldehyde fixative for 10 min, rinsed in 0.1M PBS and
ddH2O, stained with 1% cresyl violet acetate solution (Sigma Aldrich, St. Lois, MO) for 15
min at room temperature, rinsed in distilled H2O (1 min) and dehydrated in a series of 70,
95, and 100% alcohol solutions. Sections were then cleared in xylene and cover slips were
affixed with DPX mounting media (Sigma-Aldrich). Bright field images were captured at
20× magnification with a DS Fi2 color camera on a Nikon Eclipse Ni microscopy system
(Nikon Instruments Inc, Melville, NY) equipped with NIS-Elements basic research software
with large image stitching capabilities.
2.3. Agonist-stimulated [35S]GTPγS binding in vole striatal membranes
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For [35S]GTPγS binding in membranes, striata were dissected from 2 female vole brains on
ice and frozen in aliquots at −80°C. Tissue samples were thawed, homogenized with a
Tissumizer (Tekmar, Cincinnati OH) in cold TME buffer (50 mM Tris-HCl, 3 mM MgCl2, 1
mM EGTA, pH 7.4) and centrifuged at 48,000 × g for 10 min at 4°C. Pellets were
resuspended in membrane buffer and centrifuged again under identical conditions. After the
second centrifugation, pellets were homogenized in TME assay buffer (50 mM Tris-HCl, 3
mM MgCl2, 0.2 mM EGTA, 100 mM NaCl, pH 7.7). Concentration-effect curves of
agonist-stimulated [35S]GTPγS binding included 0.01–10 μM DAMGO (D-Ala2,NMePhe4,Gly-ol5]-enkephalin, Tocris Bioscience, Ellisville, MO) or U-50,488H (trans-(-)-3,4dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide HCl, Tocris
Bioscience), 30 μM GDP, 0.05 nM [35S]GTPγS (1200 Ci/mmol, Perkin Elmer, Waltham,
MA), 100 nM DPCPX (8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1 receptor
antagonist) (Moore et al., 2000), 5 μg membrane protein and TME assay buffer in a final
volume of 1 ml. Basal binding was determined in the presence of GDP and absence of drug,
and nonspecific binding was assessed in the presence of 10 μM GTPγS. Reactions were
terminated by rapid filtration under vacuum through Whatman GF/B glass fiber filters
followed by three washes with 3 ml cold 50 mM Tris-HCl buffer pH 7.7. Bound
radioactivity was determined by liquid scintillation spectrophotometry at 95% efficiency for
[35S] after overnight extraction of the filters in 4 ml ScintiSafe Econo scintillation fluid.
Data are reported as mean values ± SEM of three separate experiments, each performed in
triplicate.
2.4. Agonist-stimulated [35S]GTPγS autoradiography
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Animals were euthanized by carbon dioxide asphyxiation followed by rapid decapitation.
Brains were quickly removed and frozen in isopentane at −30 to −40°C and stored at −80°C
until sectioned for autoradiography. Coronal sections (20 μm) were obtained using a cryostat
at −22°C, and the sections were thawed onto glass slides for [35S]GTPγS autoradiography
(Sim et al., 1995). Brain sections were washed with TME buffer for 10 min at 25 °C prior to
incubation with TME assay buffer containing 2 mM GDP for 10 min at 25°C. Sections were
then incubated for two hours at 25°C in TME assay buffer containing 2 mM GDP, 100 nM
DPCPX and 0.05 nM [35S]GTPγS in the presence or absence of 3 μM of the mu agonist
DAMGO or 1 μM of the kappa agonist U50,488H (Sim-Selley et al., 1999). Separate
sections were incubated in a similar manner with the addition of 0.1 μM naloxone or 0.1 μM
nor-BNI (nor-binaltorphimine, 17,17′-(dicyclopropylmethyl)-6,6′,7,7′-6,6′-imino-7,7′Neuroscience. Author manuscript; available in PMC 2016 December 17.
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binorphinan-3,4′,14,14′-tetrol dihydrochloride, Tocris Bioscience) to inhibit mu- or kappaopioid receptors, respectively. The sections were then washed twice with 50 mM Tris-HCl,
pH 7.4 at 4 °C, rinsed once briefly with deionized water, and were exposed to phosphorimaging screens overnight. Screen images were captured with a Cyclone Phosphor Imager
(Perkin Elmer, Waltham, MA), and quantitative densitometric analysis was performed on
regions of interest using NIH ImageJ software (National Institute of Health, Bethesda, MD,
USA) in adjacent triplicate sections by an investigator that was blinded to the sex of each
subject. Regions of interest were defined by user-defined settings in NIH Image software
that selected areas of highest optical density. False color images were used for illustration
purposes, not quantification; color scales were chosen based on the highest optical density
obtained in all the brain sections, and were kept the same in all the remaining sections.
Optical densities were quantitated by comparison with [14C] brain paste standards and
values corrected to nCi/g [35S]. Data were expressed as net agonist-stimulated [35S]GTPγS
binding. Statistical comparison of net binding between male and female brains was
determined by ANOVA followed by planned comparisons within each brain region using a
post-hoc t-test with Bonferroni correction, with statistical significance indicated by a p-value
of 0.05/16 regions for DAMGO stimulation (corrected p-value of 0.0031) or 0.05/13 for
U50,488H (corrected p-value of 0.0038). All chemicals were reagent grade and purchased
from Fisher Scientific (Waltham, MA) or Sigma-Aldrich (St. Louis, MO) unless otherwise
noted.
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3. Results
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To determine the neuroanatomical distribution of mu and kappa-activated G-proteins in the
prairie vole brain, DAMGO and U50,488H were used as selective mu and kappa agonists,
respectively, to stimulate [35S]GTPγS binding in brain sections. To determine the
concentrations of agonists required to produce maximal stimulation of [35S]GTPγS,
concentration-effect curves were generated in striatal membranes prepared from female vole
brains. Results of [35S]GTPγS binding (Fig. 1) showed that DAMGO or U50,488H
stimulated binding with EC50 values of 0.15 μM or 0.098 μM, respectively. To produce
maximal stimulation, 3 μM DAMGO and 1 μM U50,488H were used in [35S]GTPγS
autoradiography experiments.
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To confirm the pharmacological specificity of these agonists in prairie vole, [35S]GTPγS
autoradiography was performed in the presence of selective mu and kappa antagonists. Fig.
2 shows representative autoradiograms from the level of forebrain and cerebellum; this
experiment was repeated in the other coronal levels used for examining [35S]GTPγS
stimulated binding with similar results (not shown). In these sections, the mu agonist
DAMGO (3 μM) produced high stimulation of [35S]GTPγS binding over basal (identical
conditions with no agonist added to the incubation buffer) in nucleus accumbens and
caudate-putamen, with moderate stimulation in cingulate cortex and in cerebellum. All of
this specific DAMGO-stimulated binding was blocked by addition of naloxone at a
concentration (0.1 μM) that is relatively mu selective, while addition of the kappa antagonist
nor-BNI (0.1 μM) had no effect on DAMGO-stimulated binding (Fig. 2, top row). Similarly,
stimulation of [35S]GTPγS binding by the kappa agonist U50,488H (1 μM) in nucleus
accumbens, caudate and cerebellum was completely blocked by nor-BNI (0.1 μM), while
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naloxone (0.1 μM) had no effect (Fig. 2, bottom row). These results confirmed that
DAMGO and U50,488H were selective in activating mu- and kappa-opioid receptor-coupled
G-proteins in prairie vole brain.
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Detailed quantitative analysis was performed on [35S]GTPγS autoradiograms of prairie vole
brains at five different coronal levels, comparing the distribution of net mu- and kappaopioid receptor stimulated [35S]GTPγS binding. Representative autoradiograms are shown
in Fig. 3; results from quantitative densitometric analysis of autoradiograms are shown in
Table 1. Representative Nissl-stained sections from prairie vole brain (Fig. 4) provide a
guide to the principal brain structures identified by [35S]GTPγS autoradiography. The
highest levels of both mu- and kappa-opioid receptor stimulated [35S]GTPγS binding
occurred in forebrain (Fig. 3, top row). For mu, high levels of activity (≥150 nCi/g net
stimulation) were observed in nucleus accumbens, caudate-putamen, and cingulate cortex,
and no activity in claustrum. In nucleus accumbens, higher levels of mu activity were
observed in core compared to shell in females. For kappa, high levels of activity were
observed in nucleus accumbens shell and (in contrast to mu activity) claustrum, with
moderate (between 150 and 95 nCi/g net stimulation) kappa activity in caudate-putamen and
nucleus accumbens core and (unlike mu) no kappa activity in cingulate cortex. In contrast to
mu activity, kappa activity in nucleus accumbens was higher in shell compared to core.
At the middle rostrocaudal level of the striatum (Fig. 3, second row), once again the highest
levels of both mu- and kappa-stimulated [35S]GTPγS binding were observed in caudateputamen. For mu, moderate activity was observed in lateral septum and ventral pallidum.
For kappa, in contrast to mu, a high level of activity was observed in ventral pallidum, but
none in lateral septum.
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At the level of the anterior diencephalon (Fig. 3, third row), mu activity was moderate in a
wide variety of structures, including amygdala, medial thalamus, and ventromedial
hypothalamus. Low levels of kappa activity (>95 nCi/g) were observed in ventromedial
hypothalamus with moderate activity observed in a thin dorsomedial band of the
hypothalamus. Such a band was not distinguishable for mu activity apart from the other
aspects of the hypothalamus. Unlike mu opioid receptor activation, low kappa-opioid
receptor activation was seen in the amygdala, medial thalamus, and ventromedial
hypothalamus.
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At the level of the posterior diencephalon (Fig. 3, fourth row), moderate levels of mu-opioid
receptor stimulated [35S]GTPγS binding were observed in central gray, hippocampus, and
interpeduncular nucleus. Low levels of mu activity were observed in medial geniculate and
superior colliculus. Low levels of kappa-opioid receptor activity were observed in central
gray and hippocampus, however unlike mu no kappa activity was observed in medial
geniculate, interpeduncular nucleus or superior colliculus.
In the hindbrain (Fig. 3, last row), low but measurable levels of mu-opioid receptor
stimulated [35S]GTPγS binding were observed in both cerebellum and parabrachial nucleus.
Low levels of kappa activity were observed in both of these regions as well, with the level of
net kappa activity in both regions barely above the minimal level of accurate quantification.
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The finding of significant DAMGO-stimulated activity in cerebellum is quite different from
rat and mouse cerebellum, where mu receptor activity is notably absent (Sim et al, 1995),
but agrees with the finding of significant mu receptor radioligand binding reported
previously in prairie vole cerebellum (Inoue et al., 2013). The finding (Fig. 2) that the
DAMGO-stimulated [35S]GTPγS is blocked specifically by naloxone confirms that this
DAMGO stimulation in cerebellum is mediated by mu receptors.
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Quantitative densitometric data was also used to determine whether there were any
significant differences between male and female for either mu- or kappa-opioid receptor
stimulated [35S]GTPγS binding in the regions examined. Table 1 shows a comparison of mu
and kappa activities in different brain regions from male and female voles. For mustimulated [35S]GTPγS binding, significant differences between male and female vole brains
were observed in only three regions: nucleus accumbens core (p<0.0001 male vs. female),
interpeduncular nucleus (p<0.0001) and hippocampus (p<0.002). In all three regions, the
levels of net mu-stimulated [35S]GTPγS binding were significantly higher in females
compared to males. In contrast, there were no significant differences between males and
females for kappa-opioid receptor stimulated [35S]GTPγS binding in any region tested.
4. Discussion
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This initial description of the neuroanatomical distribution of mu and kappa opioid receptor
G-protein activation by selective agonists suggests that interesting differences exist between
prairie voles and other rodents that typically serve as subjects for preclinical behavioral and
physiological research. One notable difference is the relatively robust activation of Gproteins by a kappa agonist, and the regions identified as being relatively more sensitive to
U50,488H are similar to those identified as having measurable amounts of kappa opioid
receptors using receptor autoradiography (Resendez et al., 2012). Kappa opioid receptors are
sparse and kappa agonists typically produce quite modest activation of G-protein signaling
as measured by [35S]GTPγS binding in both rats and mice (Hyytia et al., 1999, Slowe et al.,
1999, Park et al., 2000, Piras et al., 2010). In the prairie vole, the ability of U50,488H to
stimulate [35S]GTPγS binding was particularly robust in the accumbens shell relative to
DAMGO when compared to the above cited studies in rats and mice (Table 1). In both male
and female prairie voles, injection of nor-BNI into the shell of the accumbens inhibited
selective aggression using a resident-intruder paradigm, suggesting a key role for kappa
opioid receptors in maintaining pair bonds for both sexes (Resendez et al., 2012).
Interestingly, these investigators reported that injection of nor-BNI into the accumbens core
enhanced selective aggression only in females (Resendez et al., 2012) while in the present
study we find no differences in activation of G-proteins by a kappa agonist between male
and females, suggesting that these gender differences in the core are likely not explained by
fundamental differences in the efficiency of kappa opioid receptor-G protein coupling. This
may indicate that kappa opioid receptors are more intimately involved in reinforcement
systems in the prairie vole relatively to other typical laboratory rodent species, as has been
suggested by the involvement of kappa opioid receptors in male-male aggression following
pair bond formation (Resendez et al., 2012). The present data also suggest that the ventral
pallidum may be a region that merits exploration for involvement of kappa-opioid receptors
in prairie vole neurobiology (Table 1). The ventral pallidum is thought to coordinate motor
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responses to cognitive processes, and in the rat this region receives coordinated input from
the nucleus accumbens, amygdala, and ventral tegmental area. Stimulatory amygdala input
to the ventral pallidum is negatively modulated by dopaminergic input from the ventral
tegmental area in the rat (Maslowski-Cobuzzi and Napier, 1994). Kappa opioid agonists
decrease dopaminergic release from ventral tegmental neurons in rats via a presynaptic
action, and thereby likely serve to mitigate the influence of ventral tegmental input into this
region (Mitrovic and Napier, 2002). Kappa opioid receptors may therefore serve as a
regulator of the coordination between amygdala and ventral tegmental area influence over
motor responses and behavioral activity in the prairie vole, and the present data suggest that
the influence of kappa opioid receptors in this region may be greater than those of mu-opioid
receptors. Previous studies have shown that kappa opioid receptors in the ventral pallidum,
unlike in the accumbens, do not appear to mediate male-male aggression following pair
bond formation however (Resendez et al., 2012).
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The efficacy by which DAMGO stimulated [35S]GTPγS binding and the relative distribution
throughout the prairie vole brain was similar to that reported previously for rats and mice
(Sim et al., 1995, Hyytia et al., 1999, Park et al., 2000). The distribution of DAMGOstimulated [35S]GTPγS binding in male prairie vole brain reported here is likewise similar to
the distribution of mu-opioid receptor binding reported previously using receptor
autoradiography (Resendez et al., 2012, Inoue et al., 2013). Mu-opioid binding has also been
analyzed in female forebrain and the distribution pattern is similar to that reported here for
DAMGO-stimulated [35S]GTPγS binding, particularly comparing nucleus accumbens core
with ventral accumbens shell (Resendez et al., 2013). Unlike kappa opioid receptor
stimulation however, there were three brain regions that showed distinct differences between
male and female prairie voles for DAMGO-stimulated [35S]GTPγS binding (Table 1). These
regions were the nucleus accumbens core, interpeduncular nucleus, and hippocampus, with
females showing a greater net stimulation of [35S]GTPγS binding with DAMGO than males.
The involvement of mu-opioid receptors in the unique behavioral phenotype of pair bond
formation has been explored in the prairie vole. In females, antagonism of mu-opioid
receptors in the caudate, but not in the nucleus accumbens shell, prevents pair bond
formation (Burkett et al., 2011). When comparisons were made between the effects of muopioid antagonists on mating behavior and pair bond formation, it was shown that mu-opioid
receptors within the caudate mediate both mating and pair bonding, while antagonism of
those in the dorsomedial accumbens shell disrupt pair bond formation without affecting
mating behavior (Resendez et al., 2013). There are some discrepancies between these two
studies, however it is clear that mu opioid receptors in the dorsal striatum and dorsal
accumbens are involved in pair bond formation in female prairie voles, but not those in the
ventral accumbens shell. Similar studies have not been reported in male prairie voles,
however the present data would predict that in males the anatomical selectivity may be
relatively less pronounced than in females if mu opioid receptor G-protein activation is
predictive of in vivo pharmacology. Studies have not been reported examining the function
of mu opioid receptors in behaviors specifically related to the interpeduncular nucleus or
hippocampus in prairie voles, however the present data likewise predict that females could
potentially be more sensitive to manipulation of mu opioid activity in these regions than
males. The interpeduncular nucleus is part of the basal ganglia and is thought to mediate the
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effects of opioids on limbic function to some extent, while the hippocampus is typically
associated with learning and memory. Differences in mu opioid receptor G-protein coupling
between males and females in these regions could likewise suggest increased sensitivity to
manipulation of these behaviors by endogenous or exogenous mu agonists in female prairie
voles relative to males.
Author Manuscript
In the present study, the ability of mu and kappa opioid agonists to stimulate G-protein
activation was examined only in the monogamous prairie vole. There are a limited number
of studies demonstrating differential effects of opioids and opioid antagonists in modulating
social behaviors in prairie voles relative to non-monogamous strains such as montane voles
or meadow voles. However studies that directly compare opioid receptor densities and
localization between prairie voles and these non-monogamous vole strains have not been
documented extensively (but see Inoue et al., 2013). The present study suggests that
[35S]GTPγS autoradiography might be useful in determining if social behaviors specific to
monogamous versus non-monogamous voles correlate with the efficiency of mu and/or
kappa opioid receptor coupling to G-proteins within discrete brain regions.
Conclusion
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The distribution of mu- and kappa-opioid receptor G-protein activation in the prairie vole is
similar to that reported previously for each receptor subtype using receptor autoradiography.
Gender differences for stimulation of [35S]GTPγS binding were confined to mu-opioid
receptors, with females displaying greater activation of males in only 3 discrete brain
regions which may have important implications for gender differences in behavior that
should be explored in futures studies. Further, we validate here the autoradiographic method
for opioid receptor subtype stimulation of [35S]GTPγS binding in prairie vole brain that can
be used in future studies to determine changes in opioid receptor signaling relevant to social
behaviors as well as opioid addiction and pain management.
Acknowledgments
Funding information: Research was supported by the National Institutes of Health (Bethesda, MD) through grants
R21 NS-085533 (TJM), P51 OD-11132 (LJY and YNPRC), and P50 DA-006634 (SRC) and by Wake Forest
University Health Sciences. The funding sources had no role in study design or collection, analysis, and
interpretation of data.
Abbreviations
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DAMGO
[D-Ala2,NMe-Phe4,Gly-ol5]-enkephalin
[35S]GTPγS
guanosine 5′-O[γ-35S] triphosphate
NAc
nucleus accumbens
nor-BNI
nor-binaltorphimine
References
Ahern TH, Hammock EA, Young LJ. Parental division of labor, coordination, and the effects of family
structure on parenting in monogamous prairie voles (Microtus ochrogaster). Dev Psychobiol. 2011;
53:118–131. [PubMed: 20945408]
Neuroscience. Author manuscript; available in PMC 2016 December 17.
Martin et al.
Page 10
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Ahern TH, Young LJ. The impact of early life family structure on adult social attachment, alloparental
behavior, and the neuropeptide systems regulating affiliative behaviors in the monogamous prairie
vole (microtus ochrogaster). Front Behav Neurosci. 2009; 3:17. [PubMed: 19753327]
Breivogel CS, Childers SR, Deadwyler SA, Hampson RE, Vogt LJ, Sim-Selley LJ. Chronic delta9tetrahydrocannabinol treatment produces a time-dependent loss of cannabinoid receptors and
cannabinoid receptor-activated G proteins in rat brain. J Neurochem. 1999; 73:2447–2459.
[PubMed: 10582605]
Burkett JP, Spiegel LL, Inoue K, Murphy AZ, Young LJ. Activation of mu-opioid receptors in the
dorsal striatum is necessary for adult social attachment in monogamous prairie voles.
Neuropsychopharmacology. 2011; 36:2200–2210. [PubMed: 21734650]
Burkett JP, Young LJ. The behavioral, anatomical and pharmacological parallels between social
attachment, love and addiction. Psychopharmacology (Berl). 2012; 224:1–26. [PubMed: 22885871]
Donaldson ZR, Spiegel L, Young LJ. Central vasopressin V1a receptor activation is independently
necessary for both partner preference formation and expression in socially monogamous male
prairie voles. Behav Neurosci. 2010; 124:159–163. [PubMed: 20141291]
Hyytia P, Ingman K, Soini SL, Laitinen JT, Korpi ER. Effects of continuous opioid receptor blockade
on alcohol intake and up-regulation of opioid receptor subtype signalling in a genetic model of high
alcohol drinking. Naunyn Schmiedebergs Arch Pharmacol. 1999; 360:391–401. [PubMed:
10551276]
Inoue K, Burkett JP, Young LJ. Neuroanatomical distribution of mu-opioid receptor mRNA and
binding in monogamous prairie voles (Microtus ochrogaster) and non-monogamous meadow voles
(Microtus pennsylvanicus). Neuroscience. 2013; 244:122–133. [PubMed: 23537838]
Johnson ZV, Young LJ. Neurobiological mechanisms of social attachment and pair bonding. Current
Opinion in Behavioral Sciences. 2015; 3:38–44. [PubMed: 26146650]
Lim MM, Hammock EA, Young LJ. The role of vasopressin in the genetic and neural regulation of
monogamy. J Neuroendocrinol. 2004; 16:325–332. [PubMed: 15089970]
Maslowski-Cobuzzi RJ, Napier TC. Activation of dopaminergic neurons modulates ventral pallidal
responses evoked by amygdala stimulation. Neuroscience. 1994; 62:1103–1119. [PubMed:
7845589]
McGraw LA, Young LJ. The prairie vole: an emerging model organism for understanding the social
brain. Trends Neurosci. 2010; 33:103–109. [PubMed: 20005580]
Mitrovic I, Napier TC. Mu and kappa opioid agonists modulate ventral tegmental area input to the
ventral pallidum. Eur J Neurosci. 2002; 15:257–268. [PubMed: 11849293]
Moore RJ, Xiao R, Sim-Selley LJ, Childers SR. Agonist-stimulated [35S]GTPgammaS binding in
brain modulation by endogenous adenosine. Neuropharmacology. 2000; 39:282–289. [PubMed:
10670423]
Park Y, Ma T, Tanaka S, Jang C, Loh HH, Ko KH, Ho IK. Comparison of G-protein activation in the
brain by mu-, delta-, and kappa-opioid receptor agonists in mu-opioid receptor knockout mice.
Brain Res Bull. 2000; 52:297–302. [PubMed: 10856828]
Piras AP, Zhou Y, Schlussman SD, Ho A, Kreek MJ. Acute withdrawal from chronic escalating-dose
binge cocaine administration alters kappa opioid receptor stimulation of [35S] guanosine 5′-O[gamma-thio]triphosphate acid binding in the rat ventral tegmental area. Neuroscience. 2010;
169:751–757. [PubMed: 20452406]
Resendez SL, Dome M, Gormley G, Franco D, Nevarez N, Hamid AA, Aragona BJ. mu-Opioid
receptors within subregions of the striatum mediate pair bond formation through parallel yet
distinct reward mechanisms. J Neurosci. 2013; 33:9140–9149. [PubMed: 23699524]
Resendez SL, Kuhnmuench M, Krzywosinski T, Aragona BJ. kappa-Opioid receptors within the
nucleus accumbens shell mediate pair bond maintenance. J Neurosci. 2012; 32:6771–6784.
[PubMed: 22593047]
Sim LJ, Selley DE, Childers SR. In vitro autoradiography of receptor-activated G proteins in rat brain
by agonist-stimulated guanylyl 5′-[gamma-[35S]thio]-triphosphate binding. Proc Natl Acad Sci U
S A. 1995; 92:7242–7246. [PubMed: 7638174]
Neuroscience. Author manuscript; available in PMC 2016 December 17.
Martin et al.
Page 11
Author Manuscript
Sim-Selley LJ, Daunais JB, Porrino LJ, Childers SR. Mu and kappa1 opioid-stimulated
[35S]guanylyl-5′-O-(gamma-thio)-triphosphate binding in cynomolgus monkey brain.
Neuroscience. 1999; 94:651–662. [PubMed: 10579225]
Sim-Selley LJ, Selley DE, Vogt LJ, Childers SR, Martin TJ. Chronic heroin self-administration
desensitizes mu opioid receptor-activated G-proteins in specific regions of rat brain. J Neurosci.
2000; 20:4555–4562. [PubMed: 10844025]
Slowe SJ, Simonin F, Kieffer B, Kitchen I. Quantitative autoradiography of mu-,delta- and kappa1
opioid receptors in kappa-opioid receptor knockout mice. Brain Res. 1999; 818:335–345.
[PubMed: 10082819]
Young LJ, Lim MM, Gingrich B, Insel TR. Cellular mechanisms of social attachment. Horm Behav.
2001; 40:133–138. [PubMed: 11534973]
Young LJ, Wang Z. The neurobiology of pair bonding. Nat Neurosci. 2004; 7:1048–1054. [PubMed:
15452576]
Author Manuscript
Author Manuscript
Author Manuscript
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Martin et al.
Page 12
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Highlights
•
Opioid stimulation of [35S]GTPγS binding was validated in prairie vole brain.
•
Mu and kappa opioid stimulation was consistent with reported receptor
localization.
•
Females displayed higher mu stimulated [35S]GTPγS binding than males in 3
regions.
•
No gender differences were found for kappa stimulated [35S]GTPγS binding.
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Fig. 1. Concentration-effect curves of DAMGO and U50,488H in stimulating [35S]GTPγS
binding in striatal membranes from female prairie vole brains
Membranes were prepared and assayed for agonist-stimulated [35S]GTPγS binding as
described in Methods, using 0.01–10 μM concentrations of DAMGO and U50,488H. Results
are expressed as per cent stimulation over basal binding, and represent mean values ± SEM
of three different assays each performed in triplicate.
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Fig. 2. Pharmacological specificity of DAMGO- and U50,488H-stimulated [35S]GTPγS binding
in vole brain
Sections of male prairie vole forebrain were incubated as described in Experimental
Procedures with 0.05 nM [35S]GTPγS and 2 mM GDP with DAMGO (3 μM) as a mu
agonist or U50,488H (1 μM) as a kappa agonist, with and without the mu antagonist
naloxone (0.1 μM) or the kappa antagonist nor-BNI (0.1 μM). Similar results were obtained
at 3 additional coronal levels (not shown).
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Fig. 3. Representative autoradiograms of mu- and kappa-stimulated [35S]GTPγS binding
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Coronal sections are shown of female prairie vole brains at five brain levels (top to bottom);
forebrain, mid-striatum, anterior diencephalon, posterior diencephalon, and hindbrain.
Sections were incubated as described in Experimental Procedures with 0.05 nM [35S]GTPγS
and 2 mM GDP with 3 μM DAMGO or 1 μM U50,488H.
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Fig. 4. Nissl stained sections of prairie vole brain
Sections from female prairie vole brain are shown with identifying regional labels at 5
coronal levels.
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Table 1
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Quantitative densitometric analysis of mu- and kappa-opioid-stimulated [35S]GTPγS binding in male and
female prairie vole brain sections by autoradiography.
Net agonist-stimulated [35S]GTPγS binding, nCi/g:
Female
Region
Mu
Cingulate cortex
Kappa
168 ± 8.1
Claustrum
Male
Mu
Kappa
178 ± 6.4
168 ± 4.6
178 ± 6.9
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123 ± 5.2
240 ± 5.8*
137 ± 6.4
219 ± 8.1
168 ± 5.2
237 ± 9.2
176 ± 6.9
154 ± 5.2
99.9 ± 3.6
152 ± 4.6
106 ± 3.5
97 ± 2.9
167 ± 7.5
96.2 ± 2.5
176 ± 8.7
145 ± 4.0
76.1 ± 3.1
126 ± 2.3
80.6 ± 5.5
Nucleus accumbens core
281 ± 8.1
Nucleus accumbens shell
Caudate-putamen
Lateral septum
108 ± 5.8
Ventral pallidum
Medial thalamus
Dorsomedial hypothalamus
116 ± 5.8
106 ± 6.4
116 ± 4.6
Ventromedial hypothalamus
111 ± 3.5
61.3 ± 2.9
101 ± 3.5
74.8 ± 5.0
Amygdala
106 ± 4.6
61.3 ± 3.8
118 ± 5.8
59.6 ± 4.6
Central gray
138 ± 2.9
78.1 ± 2.5
135 ± 2.3
80.7 ± 3.3
Superior colliculus
86.7± 2.6
Medial geniculate
81.3 ± 3.4
Hippocampus
143 ± 4.6
Interpeduncular nucleus
149 ± 6.9
Cerebellum
84.7 ± 3.4
27.2 ± 1.9
89.6 ± 2.9
31.8 ± 3.9
Parabrachial nucleus
82.8 ± 4.2
39.8 ± 3.2
91.7 ± 2.0
50.3 ± 3.1
84.9 ± 3.4
79.1 ± 3.2
91.8 ± 4.2
115 ± 5.8**
76.1 ± 5.1
113 ± 5.8*
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Coronal sections from prairie vole brains (N=8, females; N=8, males) were incubated with 3 μM DAMGO (mu) or 1 μM U50,488H (kappa) with
[35S]GTPγS as described in Methods. After scanning autoradiograms on phosphor screens, quantitative densitometry was performed using NIH
Image with 14C standards (corrected for 35S) to generate data expressed as nCi/g tissue. Regions with blank data contained levels of net agoniststimulated binding that were too low (<20 nCi/g) to be accurately determined. Data are mean values ± SEM from triplicate sections from each
brain;
*
, p<0.0001 male vs. female;
**
, p=0.0014 male vs. female.
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