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Nucleus accumbens NMDA receptor activation regulates
amphetamine cross-sensitization and deltaFosB expression following
sexual experience in male rats
Lauren N. Beloate
a
,
b
, Peyton W. Weems
a
,
b
, Graham R. Casey
a
, Ian C. Webb
a
,
Lique M. Coolen
a
,
c
,
*
a
Department of Neurobiology and Anatomical Sciences, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS, 39216, USA
b
Graduate Program in Neuroscience, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS, 39216, USA
c
Department of Physiology &Biophysics, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS, 39216, USA
article info
Article history:
Received 21 August 2015
Received in revised form
14 September 2015
Accepted 16 September 2015
Available online 21 September 2015
Keywords:
Glutamate
Striatum
Sensitization
Amphetamine
FosB
cFos
abstract
Sexual experience in male rats followed by a period of absti nence causes sensitization to
D
-Amphetamine
(Amph) reward, evidenced by an increased conditioned place preference (CPP) for low doses of Amph.
Moreover, sexual experience induces neural plasticity within the nucleus accumbens (NAc), including
induction of deltaFosB, which plays a key role in Amph reward cross-sensitization. The NMDA receptor
subunit NR1 is also upregulated by mating, but the functional relevance of NMDA receptors in sex
experience-induced effects is unknown. Here, we examined the influence of intra-NAc MK 801 infusions
on sex experience-induced NAc deltaFosB and cFos expression, as well as mating- and Amph-induced
CPP in adult male rats. In experiment 1, males received MK 801 or saline into the NAc during each of
4 consecutive days of mating or handling and were tested for Amph CPP and experience-induced del-
taFosB 10 days later. Intra-NAc MK 801 during sexual behavior prevented experience-induced increases
in Amph CPP and NAc deltaFosB expression without affecting sexual behavior. In experiment 2, the ef-
fects of intra-NAc MK 801 on mating-induced CPP were examined by intra-NAc infusion of MK 801 or
saline prior to mating on conditioning days. Intra-NAc MK 801 did not affect mating-induced CPP. Next,
effects of intra-NAc MK 801 on mating-induced cFos immunoreactivity were examined. MK 801 pre-
vented mating-induced cFos expression in NAc shell and core. Together, these results provide evidence
that NAc NMDA receptor activation during sexual behavior plays a key role in mating-induced cFos and
deltaFosB expression and subsequent experience-induced cross-sensitization to Amph reward.
©2015 Published by Elsevier Ltd.
1. Introduction
Drugs of abuse cause neural plasticity within the mesolimbic
pathway, which in turn, contributes to the development and
maintenance of addiction (Chen et al., 2010; Koob and Volkow,
2010; Mameli and Luscher, 2011; Feltenstein and See, 2013;
Grueter et al., 2013; Gipson et al., 2014). The mesolimbic pathway
regulates natural reward behaviors (Frohmader et al., 2010; Olsen,
2011; Volkow et al., 2011, 2013), and repeated exposure to natural
rewards causes neural plasticity similar to that induced by drugs of
abuse (Solinas et al., 2008; Pitchers et al., 2010b, 2013, 2014; Olsen,
2011; Nader et al., 2012; Adams et al., 2013; Bardo et al., 2013),
suggesting that drugs of abuse act on the same plasticity mecha-
nisms that regulate natural reward learning (Pitchers et al., 2013,
2014). Indeed, natural reward experience influences subsequent
drug-seeking behavior in rodent models. For example, pair bonding
and environmental enrichment can serve as a protective compo-
nent to drug-seeking behavior (Aragona et al., 2007; Solinas et al.,
2008; Gipson et al., 2011; Liu et al., 2011; Burkett and Young,
2012; Puhl et al., 2012). Conversely, social isolation or the
Abbreviations: 3V, third ventricle; ac, anterior commissure; Amph,
D
-Amphet-
amine; ANOVA, analysis of variance; BLA, basolateral amygdala; CPP, conditioned
place preference; DAB, 3,3
0
-diaminobenzidine tetrahydrochloride; i.p., intraperito-
neally; MK 801, (5S,10R)-(þ)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-
5,10-imine maleate; mPFC, medial prefrontal cortex; MPN, medial preoptic nucleus;
mPOA, medial preoptic area; NAc, nucleus accumbens; PB, phosphate buffer; PBS,
phosphate buffered saline; s.c., subcutaneous.
*Corresponding author. Department of Physiology &Biophysics, University of
Mississippi Medical Center, 2500 North State Street, Jackson, MS, 39216, USA.
E-mail address: lcoolen@umc.edu (L.M. Coolen).
Contents lists available at ScienceDirect
Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm
http://dx.doi.org/10.1016/j.neuropharm.2015.09.023
0028-3908/©2015 Published by Elsevier Ltd.
Neuropharmacology 101 (2016) 154e164
removal of environmental enrichment can cause an increased
vulnerability to drug seeking (Lu et al., 2003; Raz and Berger, 2010;
Nader et al., 2012; Neisewander et al., 2012).
Repeated experience with sexual behavior causes subsequent
sensitization of drug-induced locomotor activity in female ham-
sters (Bradley and Meisel, 2001) and male rats (Pitchers et al.,
2010a, 2012). Moreover, in male rats sexual experience causes
sensitized conditioned place preference (CPP) for low doses of the
psychostimulant,
D
-Amphetamine (Amph) (Pitchers et al., 2010a),
which is dependent on a period of sexual abstinence and is long-
lasting (Pitchers et al., 2010a, 2013). Sexual experience also cau-
ses neural alterations in the nucleus accumbens (NAc) and ventral
tegmental area (VTA), including increased dendritic arborization
and spine density in NAc medium spiny neurons (Pitchers et al.,
2010a, 2013; Staffend et al., 2014), reduction of VTA dopamine
cell soma size (Pitchers et al., 2014), and upregulation of the tran-
scription factor deltaFosB, in both NAcand VTA (Meisel and Mullins,
2006; Pitchers et al., 2010b, 2013). Hence, our laboratory has pro-
posed that these neural alterations contribute to the effects of
sexual experience on cross-sensitization to Amph reward.
Glutamate is a key mediator of drug-induced neural plasticity in
the NAc (Kalivas and Volkow, 2011; Loweth et al., 2014; Pomierny-
Chamiolo et al., 2014; van Huijstee and Mansvelder, 2014). The NAc
receives multiple glutamatergic inputs, including those from the
medial prefrontal cortex (mPFC), basolateral amygdala (BLA) and
hippocampus (Britt et al., 2012; Papp et al., 2012; Tye, 2012), but a
role for glutamate in the regulation of sexual behavior or for the
effects of sexual experience on neural and behavioral plasticity has
not been established. We have previously demonstrated that sexual
experience influences the function, expression and distribution of
glutamate receptors in the NAc of male rats. In particular, patch
clamp studies showed reduced AMPA/NMDA ratios in the post-
synaptic response to frontal cortex inputs, with no alterations in
presynaptic inputs (Pitchers et al., 2012). Moreover, biochemical
analysis showed that expression of the NMDA receptor NR1 subunit
was upregulated shortly following sexual behavior, whereas AMPA
receptor GluA1 and 2 subunits were upregulated following pro-
longed periods of reward abstinence (Pitchers et al., 2012). These
results suggest an initial activation and increase in NMDA receptors
during sexual experience, followed by increased synthesis and
trafficking of AMPA receptors during the abstinence period. How-
ever, the functional relevance of NMDA receptor activation during
sexual behavior for experience-induced neuroplasticity and cross-
sensitization to Amph reward remains unknown and will be the
focus of the current set of studies.
As mentioned above, sexual experience causes long-lasting
expression of deltaFosB in the NAc (Meisel and Mullins, 2006;
Pitchers et al., 2010b, 2013). DeltafosB is also persistently
expressed in reward-related brain regions, including the NAc, in
response to drugs of abuse (Perrotti et al., 2008; Robison and
Nestler, 2011) and natural rewards, such as food (Teegarden et al.,
2009) and sucrose consumption (Wallace et al., 2008;
Christiansen et al., 2011), wheel running (Werme et al., 2002;
Greenwood et al., 2011) and environmental enrichment (Solinas
et al., 2008). DeltaFosB expression in the NAc has been shown to
play a causal role in the sensitivity to drugs of abuse, particularly
psychostimulants (Kelz et al., 1999; Nestler, 2008; Grueter et al.,
2012; Robison et al., 2013). In addition, deltaFosB expression in
the NAc is essential for the effects of sex experience on cross-
sensitization of Amph reward (Pitchers et al., 2010b, 2013) and
facilitation of sexual behavior (Pitchers et al., 2012). In the NAc,
dopamine plays a major role in the induction of deltaFosB. It is
induced in D1 receptor neurons in response to drugs of abuse (Lee
et al., 2006; Kim et al., 2009; Lobo et al., 2013), and drug- and sex-
induced deltaFosB induction is prevented by D1 receptor
antagonism (Muller and Unterwald, 2005; Pitchers et al., 2013) and
absent in D1 receptor mutant mice (Zhang et al., 2002). Besides the
role for dopamine in activation of deltaFosB, there is also limited
evidence that NMDA receptor activation may play a role, as treat-
ment with NMDA receptor agonists increases striatal deltaFosB
(Hollen et al., 1997). However, the involvement of NMDA receptor
activation in induction of this transcription factor by natural or drug
rewards is largely unexplored. Therefore, here we examine the role
of NMDA receptor activation in sex-induced deltaFosB in the NAc.
In conclusion, the current study had four objectives. First, we
tested the role of NMDA receptor activation in the NAc for the
initiation, facilitation and reward of sexual behavior in male rats.
Next, we tested the hypothesis that sex experience-induced Amph
reward cross-sensitization is dependent on NMDA receptor acti-
vation in the NAc during mating. And third, the contribution of
NMDA receptor activation for sexual experience-induced deltaFosB
was examined. In particular, the effects of intra-NAc infusions of the
NMDA receptor antagonist, MK 801, on sex behavior, experience-
induced Amph CPP and NAc deltaFosB expression were deter-
mined. Finally, as NMDA receptor activation also contributes to the
induction of cFos in the NAc by drugs of abuse (Liu et al., 1994;
Hussain et al., 2001; Yanahashi et al., 2004) and in the medial
preoptic nucleus (MPN) by sex behavior (Dominguez et al., 2007),
we tested the hypothesis that NAc NMDA receptor activity regu-
lates mating-induced cFos expression in the NAc.
2. Materials and methods
2.1. Animals
Young adult male Sprague Dawley rats (Charles River, Wil-
mington, MA; 225e250 g) were housed in same sex pairs (of
identical treatment groups) in standard Plexiglas cages. Food and
water were provided ad libitum, and animals were maintained in
temperature and humidity-controlled rooms on a 12/12 h darke-
light cycle with lights off at 6e8 am. Female Sprague Dawley rats
(Charles River; 210e225 g) were bilaterally ovariectomized and
implanted with subcutaneous (s.c.) capsules (Dow Corning tubing,
Midland, MI; 1.98 mm internal diameter) containing 5% 17-
b
-
estradiol-benzoate (in cholesterol; SigmaeAldrich, St. Louis, MO;
1cmfilled area) and received 500
m
g progesterone in 0.1 ml of
sesame oil (SigmaeAldrich; s.c.) 3e6 h prior to each mating session
to induce sexual receptivity. All experiments were carried out in
accordance with the National Institutes of Health guidelines
involving vertebrate animals in research and were approved by the
Institutional Animal Care and Use Committee at the University of
Mississippi Medical Center. All efforts were made to minimize an-
imal suffering and to reduce the number of animals used. Alter-
natives to in vivo techniques were not available.
2.2. Cannulae implantation surgeries
Each male was implanted with bilateral cannulae aimed to-
wards the NAc as described previously (Pitchers et al., 2013). Briefly,
animals were deeply anesthetized with ketamine (87 mg/ml/kg)/
xylazine (13 mg/ml/kg; i.p.) and placed into a stereotaxic apparatus
(Kopf Instruments, Tujunga, CA). Bilateral 21 gauge guide cannulae
(Plastics One, Roanoke, VA; 2.4 mm wide, 6.4 mm below pedestal)
were directed at the NAc at þ1.7 A/P and ±1.2 M/L from Bregma and
6.4 D/V from skull. Cannulae placement and drug injections were
aimed to target both NAc shell and core, as sex-induced deltaFosB
and cFos expression is comparable in both NAc subareas (Pitchers
et al., 2013). Animals received 5 mg/ml/kg carprofen (s.c.) during
surgery and 24 h later for analgesia, and were allowed to recover for
10 days prior to onset of experiments.
L.N. Beloate et al. / Neuropharmacology 101 (2016) 154e164 155
2.3. Drugs
Males received local NAc infusions of the NMDA receptor
antagonist, (5S,10R)-(þ)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]
cyclohepten-5,10-imine maleate (MK 801; Tocris, Bristol, UK Cat.
No. 0924; 1
m
g/1
m
lor1
m
g/0.5
m
l in saline), at a flow rate of 1
m
l/
min over a 1 min interval, followed byan additional 1 min with the
injection cannula (1 mm projection below guide cannula) left in
place. This dose was chosen based on previous studies showing an
effect on drug- and natural reward-related behavior (Leonibus
et al., 2002; Reynolds and KC, 2003; Wang et al., 2005; Hunt
et al., 2010; Kawasaki et al., 2011). The higher volume is based on
our previous studies and was shown to target both NAc shell and
core (Pitchers et al., 2013). Amph (
D
-Amphetamine hemisulfate
salt; SigmaeAldrich product no. A5880; 0.5 mg/ml/kg calculated on
basis of the free base; s.c.) was administered during the CPP para-
digm. At this dose, sexually experienced rats develop a CPP while
naïve rats do not (Pitchers et al., 2010a, 2013).
2.4. Sexual behavior testing
All sexual behavior testing took place 3e6 h after lights off,
under dim red lighting. Males were placed in a mating arena
(60 45 50 cm) with clean bedding for 5 min, after which they
mated with a receptive female until one ejaculation. If animals did
not reach ejaculation, tests were terminated one hour after intro-
duction of the female. Males that were assigned to sexually expe-
rienced groups mated during 4 subsequent daily sessions. Males
assigned to the sexually naïve groups were placed in identical
testing cages with clean bedding for 30 min, but did not receive
sexually receptive females. Naïve males were housed and handled
in the same rooms as the sexually experienced males and thus
subjected to distant female odors and identical noise and
disturbances.
2.5.
D
-Amphetamine conditioned place preference
CPP for Amph was conducted as previously described (Pitchers
et al., 2010a, 2013; Frohmader et al., 2011). The apparatus consists
of two main chambers separated by a smaller middle chamber, each
distinguishable by visual and tactile cues (Med Associates, St.
Albans, VT). The apparatus is unbiased, but to determine each an-
imal's initial preference, a 15 min pre-test was conducted, in which
males freely roamed the CPP apparatus. As a group, animals did not
differ in their initial preference for each of the two main chambers.
Amph or saline was paired with either CPP chamber in a counter-
balanced manner (unbiased design) during two conditioning days
(single pairing) for 30 min. The change in preference was deter-
mined during a post-test that was procedurally identical to the pre-
test. The difference in time spent in the drug-paired chamber
during the post-test minus the pre-test were calculated for each
animal and termed the CPP score.
2.6. Mating-induced conditioned place preference
CPP for mating was tested as described previously (Tenk et al.,
2009;Davis et al., 2010; Di Sebastiano et al., 2011; Frohmader
et al., 2011) and the procedure was similar to that for Amph CPP.
A 15 min pre-test determined the initial preference for each animal.
As a group, the animals showed no preference. The sex-paired
chamber was assigned to the initially non-preferred chamber
(biased design). During conditioning, males were placed into the
sex-paired chamber (single pairing) immediately after ejaculation
for 30 min. On the alternate conditioning day, animals were placed
into the non sex-paired chamber for 30 min. The order of the two
conditioning days was counterbalanced. Preference scores (% time
spent in sex-paired chamber) and difference scores (time spent in
paired unpaired chamber) were calculated for each animal.
2.7. Immunohistochemistry
2.7.1. Tissue preparation
Animals received an overdose of sodium pentobarbital (Vortech
Pharmaceutical Ltd., Dearborn, MI; 390 mg/ml/kg; i.p.) and were
transcardially perfused with 10 ml saline (0.9% NaCl (Sigma-
eAldrich) in ddH
2
O) and 500 ml of 4% paraformaldehyde (Electron
Microscopy Sciences, Hatfield, PA; in 0.1 M phosphate buffer (PB)).
The brains were removed and post-fixed for one hour in the same
fixative at room temperature and stored in a sucrose solution
(Fisher Scientific, Fair Lawn, NJ; 20% in 0.1 M PB containing 0.01%
sodium azide (SigmaeAldrich); at 4
C). Brains were sectioned
coronally (35
m
m) into 4 parallel series, using a freezing microtome
(SM 2000R, Leica Biosystems, Lawrenceville, GA) and stored
in 20
C in cryoprotectant solution (30% sucrose in 0.1 m PB
containing 30% ethylene glycol (Fisher Scientific) and 0.01% sodium
azide).
2.7.2. DeltaFosB and cFos immunoprocessing
Free floating sections were thoroughly washed in phosphate-
buffered saline (PBS; pH 7.4) at room temperature under gentile
agitation between each incubation. Tissue was exposed to 1% H
2
O
2
for 10 min and incubation solution consisting of PBS containing
0.1% bovine serum albumin (Fisher Scientific) and 0.4% Triton X-100
(SigmaeAldrich) for 1 h, followed by incubation in either pan-FosB
rabbit polyclonal antibody raised against an internal region shared
by FosB and deltaFosB (Santa Cruz Biotechnology, Santa Cruz, CA;
sc-48; 1:5000 in incubation solution) or rabbit anti-cFos (Santa
Cruz Biotechnology; sc-52; 1:2500 in incubation solution) for 17 h.
Sections were then incubated in biotinylated goat anti-rabbit IgG
(Vector Laboratories, Burlingame, CA; 1:500 in incubation solution;
1 h), avidinebiotin-horseradish peroxidase (ABC Elite; Vector
Laboratories; 1:1000 in PBS; 1 h) and 0.02% 3,3
0
-diaminobenzidine
tetrahydrochloride (DAB; SigmaeAldrich) with 0.02% nickel sulfate
(SigmaeAldrich) and 0.015% H
2
O
2
in 0.1 M PB (10 min). Sections
were mounted onto Superfrost plus glass slides (Fisher Labora-
tories), dehydrated and coverslipped with dibutyl phthalate xylene
(Electron Microscopy Sciences). Both primary antibodies have been
previously validated to specifically visualize deltaFosB or cFos un-
der these conditions (Perrotti et al., 2004, 2008; Di Sebastiano et al.,
2010, 2011; Pitchers et al. 2010b, 2013).
2.7.3. Cannulae placement verification
The placement of cannulae was confirmed using sections
stained for cFos or deltaFosB. Only animals with correct placement
were included in analyses. For clarity, final numbers of animals for
each of the experiments are listed in the appropriate figure legends.
3. Experimental design
3.1. Experiment 1: effects of intra-NAc MK 801 on sexual behavior
and experience-induced Amph CPP and NAc deltaFosB expression
The main goals of this study were to investigate if intra-NAc MK
801 during mating experience would prevent experience-induced
Amph CPP sensitization and NAc deltaFosB expression.
Sexually experienced and naïve groups of males received intra-
NAc MK 801 (1
m
g in 0.5
m
lor1
m
l saline) or saline 15 (1
m
l) min prior
to each of the four daily mating or handling sessions. Latencies to
first mount, intromission and ejaculation were analyzed between
days 1 and 4 for all groups using a two-way analysis of variance
L.N. Beloate et al. / Neuropharmacology 101 (2016) 154e16 4156
(ANOVA; drug day) and StudenteNewmaneKeuls post-hoc test.
Latencies to first mount, intromission and ejaculation between days
1 and 4 were also analyzed within each group using a one-tailed
ManneWhitney rank sum test. 95% confidence levels were used
for all tests. Animals were included in analyses only for the sexual
parameters displayed for each of the sessions.
Ten days following the last mating or handling session, males
were tested for Amph CPP. CPP scores (time spent in the paired
chamber during the post-test minus the pre-test) were compared
between groups and analyzed using a two-way ANOVA (drug sex)
and HolmeSidak post-hoc tests. 95% confidence levels were used for
all tests. Animals were excluded from analysis if they displayed an
initial preference of 120 s or more forone of the 3 chambers, had CPP
scores that were more than 2 standard deviations away from the
group mean, did not reach three or more days with one ejaculation
during sexual experience, or had missed cannula placements.
One day after the Amph CPP post-test, brains were collected and
analyzed for deltaFosB. DeltaFosB-immunoreactive cells were
quantified in the NAc shell and core using a standard area of
analysis (400 600
m
m per subregion) on a light microscope with a
drawing tube attachment (Leica DMR; Leica Microsystems, Inc.,
Buffalo Grove, IL) as described previously (Pitchers et al., 2010b,
2013). Briefly, at least two NAc sections per subregion, per animal
were counted and averaged, then a group average was calculated.
Dorsal striatum (200 600
m
m area of analysis) immuno-positive
cells were counted to check for drug spread. Group averages were
compared between groups using a two-way ANOVA (drug sex
experience) and HolmeSidak post-hoc tests. 95% confidence levels
were used for all tests.
3.2. Experiment 2: effects of intra-NAc MK 801 on mating-induced
CPP and NAc cFos expression
The main goals of this study were to investigate if intra-NAc MK
801 during mating would prevent sexual reward and mating-
induced neuronal activation in the NAc. Males received intra-NAc
MK 801 (1
m
g in 0.5
m
lor1
m
l saline) or saline 15 min prior to
introduction of female on the sex conditioning day for CPP. La-
tencies to first mount, intromission and ejaculation were recorded
and compared between groups using a one-way ANOVA on ranks.
CPP preference and difference scores were compared between pre-
and post-tests, within groups, using unpaired t-tests. 95% confi-
dence levels were used for all tests. Animals were excluded if they
spent more than 120 s in one of the three chambers.
One day following the sex CPP post-test, the same males
received intra-NAc MK 801 (1
m
g in 0.5
m
lor1
m
l saline) or saline
and either mated or were placed in the cages without females. One
hour after introduction of females, brains were collected and
analyzed for cFos expression. cFos immunoreactive cells were
quantified in the NAc shell and core with a standard area of analysis
(400 600
m
m per subregion) on a light microscope with a drawing
tube attachment (Leica DMR) as described in Experiment 1. At least
two NAc sections per subregion per animal were counted and
averaged, then a group average was calculated. medial preoptic
nucleus (MPN) (600 800
m
m area of analysis) immuno-positive
cells were counted to check for drug spread. The no mating
groups did not differ between drug treatments on any measures
and were combined for analysis. Groups were compared using a
one-way ANOVA and Fisher LSD post hoc test, with 95% confidence
levels.
4. Results
4.1. Intra-NAc MK 801 did not disrupt sexual behavior
In the first experiment, intra-NAc infusions of MK 801 did not
affect sexual behavior on any of the four daily mating tests, and no
differences between groups were detected. Moreover, animals in all
groups showed facilitation of sexual behavior with experience
(mount latencies: F
(2,92)
¼5.413, p ¼0.022; intromission latencies:
F
(2,91)
¼6.742, p ¼0.011; ejaculation latencies: F
(2,91)
¼8.565,
p¼0.004), evidenced by significantly lower latencies or trends in
the fourth mating session compared to the first mating session to
mount, intromit, and ejaculate in all groups (Fig. 1AeC; p <0.05).
Thus, blockade of NMDA receptors in the NAc did not disrupt
initiation or performance of sexual behavior or the experience-
induced facilitation of sexual behavior.
4.2. Intra-NAc MK 801 prevented sex experience-induced Amph CPP
One week following sexual experience, all groups were tested
for Amph CPP (Fig. 1E). There was an overall interaction effect of
sexual experience and MK 801 treatment (F
(2,58)
¼7.774, p ¼0.001).
Specifically, sexually experienced control males that received intra-
NAc vehicle injections displayed a significantly increased CPP score
compared to vehicle-treated naïve controls (p ¼0.042), confirming
our previous findings (Pitchers et al., 2010a, 2013). Intra-NAc in-
fusions of MK 801 during mating experience prevented this effect.
CPP scores in both of the MK 801-injected sexually experienced
groups did not differ from vehicle-treated naïve males, and CPP
scores of animals injected with 1
m
l MK 801 were significantly
lower than vehicle-treated experienced males (p ¼0.004). Intra-
NAc infusions of MK 801 in sexually naïve controls did not affect
Amph CPP at the lower volume (0.5
m
l). However, infusions of the
higher volume (1
m
l) of MK 801 in sexually naïve males caused a
significantly increased CPP score compared to vehicle-infused
sexually naïve males (p ¼0.016). This finding is in agreement
with effects of systemic MK 801 on psychostimulant sensitization
(Rung et al., 2005; Eyjolfsson et al., 2006; Landa et al., 2014), but in
sharp contrast to the effect of NMDA receptor blockade in sexually
experienced males in the current study.
4.3. Intra-NAc MK 801 blocked sex experience-induced deltaFosB
There were significant main effects of sexual experience (core:
F
(1,40)
¼31.357, p <0.001; shell: F
(1,38)
¼19.129, p <0.001) and MK
801 treatment (core: F
(2,40)
¼13.660, p <0.001; shell:
F
(2,38)
¼16.532, p <0.001) and a significant interaction effect (core:
F
(2,40)
¼20.949, p <0.001; shell: F
(2,38)
¼18.498, p <0.001) on NAc
deltaFosB. Sexually experienced vehicle-treated males had signifi-
cantly more deltaFosB immunoreactive cells in the NAc core
(Fig. 2G) and shell (Fig. 2H) compared to naïve saline-treated con-
trols (p <0.001), as reported previously (Pitchers et al., 2010b,
2013). Intra-NAc MK 801 injections prior to each mating session
prevented this effect. Numbers of deltaFosB cells in both MK 801-
infused sexually experienced groups did not differ from naïve
controls and were significantly lower compared to vehicle-treated
sexually experienced males (p <0.001). Intra-NAc MK 801 did
not affect deltaFosB expression in naïve males. Finally, the MK 801
infusions appeared to be restricted to the NAc and did not reduce
sex experience-induced deltaFosB expression in the dorsal stria-
tum. Specifically, in the medial caudate putamen, an area imme-
diately dorsal to the NAc, both sexually experienced vehicle- and
1
m
l MK 801-treated groups had significantly higher numbers of
deltaFosB compared to vehicle-treated naïve males and did not
differ from each other (vehicle naive: 5.714 ±4.071; vehicle
L.N. Beloate et al. / Neuropharmacology 101 (2016) 154e164 157
experienced: 34.40 ±8.38, p <0.001; 1
m
l MK 801 experienced:
38.67 ±9.39, p <0.001).
4.4. Intra-NAc MK 801 did not prevent mating-induced CPP
The second experiment replicated the finding that intra-NAc MK
801 does not affect the initiation or performance of sexual behavior
(Table 1). Moreover, MK 801 did not prevent mating-induced CPP
(Fig. 3). Mating induced a similar CPP in MK 801- and vehicle-
treated groups, indicated by significantly increased preference
(percentage of time in the sex-paired chamber) and difference
scores (time in paired minus unpaired chamber) during the post-
test compared to pre-test (p <0.05). The 0.5
m
l MK 801-injected
group had a trend towards higher preference score during the
Fig. 1. Intra-NAc MK 801 blocks sexual experience-induced Amph CPP but does not affect sexual behavior. (AeC) Quantitative analysis of latencies (in seconds) to first mount (A),
first intromission (B) or ejaculation (C) on days 1 and 4 of consecutive days of mating for groups receiving intra-NAc saline (white bars,n¼20), 0.5
m
lMK801(gray bars,n¼8) or
1
m
lMK801(black bars,n¼22). Data represent mean ±SEM. * Denotes significant difference within group compared to day 1: A) mount latencies: vehicle: p ¼0.040, 0.5
m
lMK801:
p<0.001,1
m
lMK801:p¼0.06; B) intromission latencies: vehicle: p ¼0.043, 0.5
m
lMK801:p<0.0 01, 1
m
lMK801:p¼0.066; C) ejaculation latencies: vehicle: p ¼0.002, 0.5
m
lMK
801: p ¼0.095, 1
m
lMK801:p¼0.021; D) Coronal NAc sections indicating injection sites. Cannulae were placed bilaterally, but are shown unilaterally for easier viewing. Left side:
sexually naïve males (dotted outline); Right side: sexually experienced males (solid outline). White fill: saline; Light gray fill: 0.5
m
lMK801;Dark gray fill:1
m
lMK801.(E) CPP scores,
defined as the time spent in Amph-paired chamber during post-test minus pre-test, for sexually naïve (naive) and sexually experienced (experienced) groups that received intra-NAc
saline (white bars, naïve n ¼15, exp n ¼14), 0.5
m
lMK801(gray bars, naïve n ¼7, exp n ¼7) or 1
m
lMK801(black bars, naïve n ¼7, ex p n ¼14) during mating or handling. Data are
presented as mean ±SEM. * Indicates significant difference compared to saline naïve group; # indicates significant difference compared to all other sexually experienced groups.
L.N. Beloate et al. / Neuropharmacology 101 (2016) 154e16 4158
post-test compared to pre-test, although this increase did not reach
significance (p ¼0.055).
4.5. Intra-NAc MK 801 blocked mating-induced NAc cFos
expression
Finally, intra-NAc MK 801 blocked mating-induced cFos
expression. There was a significant interaction effect of sexual
behavior and MK 801 (F
(3,24)
¼49.196, p <0.0 01). In vehicle-treated
control animals, mating increased cFos expression in the NAc core
(Fig. 4E) and shell (Fig. 4F), compared to non-mating controls
(p <0.001). Both volumes of MK 801 prevented mating-induced
cFos in the NAc shell and core, as numbers of cFos cells in MK
801 groups did not differ from non-mating controls and were
significantly lower compared to vehicle-treated mating males
(Fig. 4F; p 0.001). Again, intra-NAc MK 801 infusions did not
affect mating-induced cFos expression in nearby brain regions. In
particular, the was analyzed, as mating induces robust cFos in this
region (Veening and Coolen, 2014) and the MPN is located caudal to
the NAc and close to the ventricle. Indeed, in the MPN, there was a
main effect of sex behavior (F
(3,14)
¼36.725, p <0.001) but not of
MK 801 treatment, and mating-induced cFos in the MPN inde-
pendent of the intra-NAc drug infusion (Fig. 5).
5. Discussion
The current study demonstrates that NMDA receptor activation
in the NAc during sexual behavior is critical for the cross-sensitizing
effects of sexual experience on Amph reward as intra-NAc MK 801
injections during sexual experience prevented increased CPP for
Amph. Moreover, NAc NMDA receptor activation during sexual
behavior contributes to mating-induced cFos and deltaFosB
expression but not to sexual behavior or reward. In summary, NAc
NMDA receptor activation during sexual behavior regulates
mating-induced neural activation, neural alterations and behav-
ioral plasticity following sexual experience, but is not critical for the
initiation, expression, facilitation or reward of sex behavior.
Sexual experience causes cross-sensitization of CPP for Amph
after a period of abstinence from sexual reward (Pitchers et al.,
2010a, 2010b, 2013). Moreover, sexual experience upregulates
NR1 NMDA receptor subunit expression in the NAc (Pitchers et al.,
2012), suggesting that NMDA receptors are activated during sexual
experience and mediate these cross-sensitizing effects. Indeed, the
present study shows that activation of NMDA receptors in the NAc
during the acquisition of sexual experience is critical for
experience-induced cross-sensitization of Amph CPP. In previous
studies, NAc NMDA receptor blockade has been shown to block
Fig. 2. Intra-NAc MK 801 blocks sex experience-induced NAc deltaFosB expression.
(AeF), Representative images of NAc deltaFosB expression in sexually naïve and
experienced males that received intra-NAc saline (A,B), 0.5
m
lMK801(C,D)or1
m
lMK
801 (E,F). Scale bar indicates 100
m
m. Quantification of deltaFosB-positive neurons in
NAc core (G) and shell (H): saline: naïve, n ¼15; experienced, n ¼14; 0.5
m
lMK801:
naïve, n ¼7; experienced, or 1
m
l MK 801: naïve, n ¼7; exp, n ¼14. Data are presented
as mean ±SEM. * Indicates significant difference compared to saline naïve (all
p<0.001).
Table 1
Sexual behavior parameters following intra-NAc MK 801. Latencies (in seconds) to
first mount, first intromission or ejaculation during the single mating session con-
ducted during the CPP conditioning trial (sal, n ¼6; 0.5
m
l MK, n ¼9; 1
m
l MK, n ¼7)
and single mating session conducted to investigate mating-induced cFos expression
(sal, n ¼5; 0.5
m
l MK, n ¼5; 1
m
l MK, n ¼5). Data represent mean ±SEM. No sig-
nificant differences between groups were detected.
Latencies (s)
Mount Intromission Ejaculation
CPP conditioning
Saline 593.3 ±291.6 599 ±289.5 961 ±167.3
MK 801 0.5
m
l 318.6 ±152.8 394.4 ±205.7 955 ±249.4
MK 801 1
m
l 231 ±55.1 296.1 ±73.6 852.6 ±135.5
Final mating
Saline 99.4 ±43.8 157.8 ±74.7 708.3 ±110.9
MK 801 0.5
m
l 115.8 ±35.6 126.4 ±31.6 975.5 ±166.8
MK 801 1
m
l 73.2 ±11.4 73.8 ±11.9 1467.5 ±652.6
L.N. Beloate et al. / Neuropharmacology 101 (2016) 154e164 159
morphine- (Popik and Kolasiewicz, 1999; Ma et al., 2006; Kao et al.,
2011; Xu et al., 2012) and ethanol-induced CPP (Gremel and
Cunningham, 2009) but the blockade took place during condi-
tioning or reconsolidation of the drug CPP. NMDA receptors in the
NAc have been shown to regulate the development of psychosti-
mulant locomotor sensitization (Wolf, 1998; Vanderschuren and
Kalivas, 2000). Specifically, cocaine exposure leads to AMPA re-
ceptor current changes on NAc medium spiny neurons that are
dependent on NMDA receptor activation (Ungless et al., 2001), and
Amph behavioral sensitization is prevented by co-administration
(systemic) of MK 801 (Wolf and Jeziorski, 1993). Sex behavior also
results in locomotor sensitization by Amph (Pitchers et al., 2010a,
2012) and methamphetamine (Frohmader et al., 2011), but it is
Fig. 3. Intra-NAc MK 801 does not affect mating-induced CPP. A, Coronal NAc sections
indicating injection sites, represented unilaterally (dotted outline, on left: no mating on
last mating day, solid, on right: mating; white fill: saline; light gray: 0.5
m
l MK; dark gray:
1
m
l MK). The preference scores (percentage of time in the sex-paired chamber) (B) and
difference scores (time in paired eunpaired chamber) (C) during the pre-test and the
post-test within groups of animals that received intra-NAc saline (white bars,n¼6),
0.5
m
lMK801(gray bars,n¼9) or 1
m
lMK801(black bars,n¼10) prior to mating
during conditioning (preference scores: vehicle: p ¼0.048; difference scores: vehicle:
p¼0.038; 0.5
m
lMK801:p¼0.034; 1
m
lMK801:p¼0.033). Data are presented as
mean ±SEM. * Denotes significant difference within group during post-test compared
to pre-test.
Fig. 4. Intra-NAc MK 801 blocks mating-induced NAc cFos expression. (AeD), Repre-
sentative images of NAc cFos-positive neurons from males that received intra-NAc
saline (B), 0.5
m
lMK801(C)or1
m
lMK801(D) prior to mating (þmating)or
handling (no mating)(A). Scale bar indicates 100
m
mac¼anterior commissure.
Quantitative data of cFos expression in NAc core (E) and shell (F) from males that
received intra-NAc saline (light gray bars,n¼5), 0.5
m
lMK801(dark gray bars,n¼6)
or 1
m
lMK801(black bars,n¼5) prior to mating or no mating (white bars,n¼12; 4
from each drug group). Data are presented as mean ±SEM. * Indicates significant
difference compared to no mating group (core: 0.5
m
lMK801:p<0.001, 1
m
lMK801:
p<0.001; shell: 0.5
m
lMK801:p<0.0 01, 1
m
lMK801:p¼0.0 01); # shows significant
difference compared to saline þmating group (p <0.001).
L.N. Beloate et al. / Neuropharmacology 101 (2016) 154e16 4160
unknown if NMDA receptor activation during sex experience is
required for this effect.
In addition, the current results demonstrate that sexual
experience-induced deltaFosB expression in the NAc is dependent
on NMDA receptor activation during mating. We have previously
shown that functional blockade of deltaFosB in the NAc, by over-
expression of the dominant-negative binding partner JunD, pre-
vented the cross-sensitization to Amph CPP following sexual
experience and abstinence (Pitchers et al., 2013). Hence, the effects
of NMDA receptor blockade on attenuation of Amph CPP sensiti-
zation may be mediated via blockade of deltaFosB expression
during the mating session. Drug- and natural reward-induced
deltaFosB expression has been shown to be expressed in D1 re-
ceptor neurons and is dependent on D1 receptor activation (Zhang
et al., 2002; Muller and Unterwald, 2005; Lobo et al., 2013). In
particular, our lab has shown that NAc D1 dopamine receptor
activation is required for sex experience-induced cross-sensitiza-
tion to Amph CPP (Pitchers et al., 2013). The current findings show
that in addition to the role of dopamine receptor activation, NMDA
receptor activation is also functionally involved in sex experience-
induced deltaFosB upregulation in the NAc. Although there is
currently no direct evidence for glutamate release in the NAc dur-
ing mating, is it well established that dopamine is released in the
NAc during mating (Pfaus et al., 1990; Meisel et al., 1993; Fumero
et al., 1994) and that dopamine and glutamate are co-released in
mesoaccumbens axons (Tecuapetla et al., 2010; Zhang et al., 2015).
Together these findings suggest a potential interaction between
NMDA and D1 receptor activation during reward experience. In
agreement, Amph locomotor and CPP sensitization is prevented by
NMDA gene deletion in D1-expressing medium spiny neurons
(Beutler et al., 2011), and D1 and NMDA receptors form direct in-
teractions in the striatum, and other areas, such as cortex and
hippocampus (Lee et al., 2002; Ladepeche et al., 2014). In the
striatum NMDA activity upregulates D1 receptor internalization
and trafficking (Scott et al., 2002, 20 06), and D1 activation increases
glutamate transmission (Harvey and Lacey, 1997) and NMDA Ca
2þ
signaling and ERK activation (Cahill et al., 2014).
A third finding of the current study was that mating-induced
cFos expression in the NAc is dependent on NMDA receptor acti-
vation. cFos is an immediate early gene commonly used as a marker
for neuronal activation and is upregulated in the NAc in responseto
drugs of abuse (Hope et al., 1992; Persico et al., 1993; Steiner and
Gerfen, 1993) and sexual behavior (Veening and Coolen, 2014).
We had previously shown that activation of NMDA receptors is
critical for mating-induced cFos in the medial preoptic area (mPOA)
(Dominguez et al., 2007). In this brain area, extra-synaptic levels of
glutamate are elevated during sexual behavior (Dominguez et al.,
2006, 2007); mating leads to phosphorylation of NMDA re-
ceptors; and intra-mPOA MK 801 blocks mating-induced phos-
phorylation of NMDA and cFos immunoreactivity (Dominguez
et al., 2007). cFos has been shown to regulate cocaine behavioral
sensitization and cocaine-induced deltaFosB in the striatum, spe-
cifically in D1 receptor-expressing neurons (Zhang et al., 2006) but
the role of cFos induction in the NAc for the expression of sexual
behavior remains unknown. The current study suggests that sex-
induced cFos in the NAc regulates sex experience-induced delta-
FosB expression and cross-sensitization of Amph CPP.
Results of the present study demonstrate that activation of
NMDA receptors in the NAc is not required for initiation and
expression of sexual behavior, or sexual reward as determined by
CPP. This is in apparent contrast to the effects of excitotoxic lesions
of the NAc which disrupt initiation and expression of sexual
behavior in male rats, but not the preference for receptive females
(Kippin et al., 2004). In addition, the NAc is generally thought to be
critical for learning of stimulus-reward associations and mediating
goal-directed behavior (Robinson and Kolb, 2004; Richard et al.,
2013). Hence, our results indicate that NMDA receptor activation
in the NAc during mating is not required for these functions, similar
to the lack of involvement of NAc mGluR5 (Pitchers et al., 2015,
unpublished observations) and dopamine D1 receptors (Pitchers
et al., 2013). Likewise, intra-NAc injections of NMDA antagonists
block the acquisition of food-reinforced instrumental learning
(Kelley et al., 1997; Doerks et al., 2002; Kelley, 2004), but do not
block food intake (Reynolds and KC, 2003). Instead, a role for AMPA
receptor activation in motivation for food intake has been shown
(Reynolds and KC, 2003), suggesting that glutamate acts in the NAc
to mediate goal-directed learning and behavior, but via activation
of AMPA receptors. The present results also indicate that mating-
induced CPP was unaffected by intra-NAc MK 801 during the con-
ditioning day. NAc NMDA receptor antagonism has been shown to
block drug-induced CPP (Popik and Kolasiewicz, 1999; Ma et al.,
2006; Gremel and Cunningham, 2009; Kao et al., 2011; Xu et al.,
2012), without affecting social interaction- or food-induced CPP
(Ma et al., 2006). Together, these results suggests a role for NAc
NMDA receptors primarily in drug reward or cross-sensitization to
drug reward, but not of associated learning and CPP expression for
natural rewards, including sexual behavior.
Finally, the current results demonstratedthat repeated infusions
of MK 801 into the NAc caused a sensitization to Amph CPP in naïve
rats 10 days after final infusion. It is well established that systemic
administration of MK 801 leads to Amph locomotor sensitization in
Fig. 5. Intra-NAc MK 801 does not affect mating-induced MPN cFos expression. (AeD)
Representative images of cFos-positive neurons in MPN from males that received intra-
NAc saline (B), 0.5
m
lMK801(C)or1
m
lMK801(D) prior to mating (þmating)or
handling (no mating)(A). 3V ¼third ventricle. Scale bar indicates 100
m
m. (E) Quan-
tification of MPN cFos-positive neurons in the same animals as in Fig. 4. Data are
presented as mean ±SEM. * Indicates significant difference compared to no mating
group (all p <0.001).
L.N. Beloate et al. / Neuropharmacology 101 (2016) 154e164 161
rats (Rung et al., 2005; Eyjolfsson et al., 2006; Landa et al., 2014) but
its effects on cross-sensitization to drug CPP has not been
demonstrated. Therefore, the current findings expand on this
knowledge by showing that MK 801 results in sensitization of
Amph reward, and specifically by acting in the NAc. It is of note that
this effect of MK 801 in sexually naïve animals caused an opposite
effect to that in mating animals; i.e. a reward sensitization in
sexually naïve males instead of a blockade of cross-sensitization
following sexual experience. This may be explained by the ac-
tions of MK 801 in the absence of the endogenous ligand for NMDA
receptors. Indeed, studies suggest that MK 801's actions depend on
the activity state of NMDA receptors (Huettner and Bean, 1988;
Yuzaki et al., 1990; Reynolds and Miller, 1998). Furthermore, cell
culture studies have shown that MK 801 binding to the NMDA re-
ceptor is long lasting in the absence of glutamate or Mg
2þ
, while
dissociation of the drug from the receptor is greatly enhanced by
the presence of Mg
2þ
and opening of the channel (McKay et al.,
2013). Hence, the pharmacokinetics of MK 801 action may have
drastically differed in mating versus naïve animals.
5.1. Conclusions
In conclusion, the current study provides evidence for NAc
NMDA receptor regulation of mating-induced deltaFosB and cFos
and sexual experience-induced psychostimulant reward cross-
sensitization. Together, the results suggest that NAc NMDA recep-
tor activation during mating regulates the long-term effects of
natural reward experience via induction of cFos and deltaFosB.
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