ΔJunD overexpression in the nucleus accumbens prevents sexual reward in female Syrian hamsters

Abstract

Motivated behaviors, including sexual experience, activate the mesolimbic dopamine system and produce long-lasting molecular and structural changes in the nucleus accumbens. The transcription factor ΔFosB is hypothesized to partly mediate this experience-dependent plasticity. Previous research in our laboratory has demonstrated that overexpressing ΔFosB in the nucleus accumbens of female Syrian hamsters augments the ability of sexual experience to cause the formation of a conditioned place preference. It is unknown, however, whether ΔFosB-mediated transcription in the nucleus accumbens is required for the behavioral consequences of sexual reward. We therefore used an adeno-associated virus to overexpress ΔJunD, a dominant negative binding partner of ΔFosB that decreases ΔFosB-mediated transcription by competitively heterodimerizing with ΔFosB before binding at promotor regions on target genes, in the nucleus accumbens. We found that overexpression of ΔJunD prevented the formation of a conditioned place preference following repeated sexual experiences. These data, when coupled with our previous findings, suggest that ∆FosB is both necessary and sufficient for behavioral plasticity following sexual experience. Furthermore, these results contribute to an important and growing body of literature demonstrating the necessity of endogenous ΔFosB expression in the nucleus accumbens for adaptive responding to naturally rewarding stimuli.

Full text

Delta JunD overexpression in the nucleus accumbens prevents
sexual reward in female Syrian hamsters
Laura E. Beena, Valerie L. Hedgesa, Vincent Vialoub, Eric J. Nestlerc, and Robert L. Meisela
aUniversity of Minnesota, Department of Neuroscience, 6-145 Jackson Hall, 321 Church Street,
Minneapolis, MN 55455, USA
bINSERM, U952, CNRS, UMR7224, UPMC, Paris, 75005, France
cFishberg Department of Neuroscience and Friedman Brain Institute, Mount Sinai School of
Medicine, New York, New York 10029, USA
Abstract
Motivated behaviors, including sexual experience, activate the mesolimbic dopamine system and
produce long-lasting molecular and structural changes in the nucleus accumbens. The transcription
factor ΔFosB is hypothesized to partly mediate this experience-dependent plasticity. Previous
research in our laboratory has demonstrated that overexpressing ΔFosB in the nucleus accumbens
of female Syrian hamsters augments the ability of sexual experience to cause the formation of a
conditioned place preference. It is unknown, however, whether ΔFosB-mediated transcription in
the nucleus accumbens is required for the behavioral consequences of sexual reward. We therefore
used an adeno-associated virus to overexpress ΔJunD, a dominant negative binding partner of
ΔFosB that decreases ΔFosB-mediated transcription by competitively heterodimerizing with
ΔFosB before binding at promotor regions on target genes, in the nucleus accumbens. We found
that overexpression of ΔJunD prevented the formation of a conditioned place preference following
repeated sexual experiences. These data, when coupled with our previous findings, suggest that
ΔFosB is both necessary and sufficient for behavioral plasticity following sexual experience.
Furthermore, these results contribute to an important and growing body of literature demonstrating
the necessity of endogenous ΔFosB expression in the nucleus accumbens for adaptive responding
to naturally rewarding stimuli.
Keywords
Delta FosB; Sex; Conditioned Place Preference; Adeno-Associated Virus; Plasticity
INTRODUCTION
Plasticity in the striatum, like many areas of the brain, is critical for adaptive responses to
environmental stimuli at the molecular, cellular, and behavioral levels. Motivated behaviors,
such as sex (Meisel & Mullins, 2006, Pitchers et al., 2010b, Wallace et al., 2008),
aggression (Couppis & Kennedy, 2008, Staffend & Meisel, 2012), and wheel running
(Greenwood et al., 2011, Vargas-Perez et al., 2003, Werme et al., 2002), as well as chronic
exposure to drugs of abuse (Chen et al., 2010, Robinson & Kolb, 2004, Russo et al., 2010),
result in activation of the mesolimbic dopamine system and long-lasting changes in the
nucleus accumbens (NAc). Structural changes, particularly the formation of dendritic spines,
are important components of this experience-based plasticity, which persists long after the
Corresponding Author: Laura Been, lbeen@umn.edu, phone: 612-625-0784, fax: 612-626-9201.
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Genes Brain Behav. 2013 August ; 12(6): 666–672. doi:10.1111/gbb.12060.
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motivated behavior or drug administration has ceased (Meisel & Mullins, 2006, Pitchers et
al., 2010b, Robinson & Kolb, 2004).
Although the intervening molecular events that cause changes in dendritic morphology have
not been fully described, the transcription factor ΔFosB appears to be at the heart of the
molecular pathway that mediates the enduring structural and behavioral changes that occur
following experience with motivated behaviors and/or drugs of abuse (Nestler, 2008).
ΔFosB is an alternative splice product of the immediate early gene fosB and shares
homology with other Fos family transcription factors, which heterodimerize with Jun family
proteins to activate AP-1-mediated gene transcription (Robison & Nestler, 2011). Unlike
other Fos family proteins that are highly unstable and return to basal levels within hours of
stimulation, ΔFosB accumulates following repeated stimulation and remains elevated and
stable for extended periods of time (Chen et al., 1997, Nestler, 2008, Robison & Nestler,
2011). This stability is a hypothesized mechanism by which changes in gene expression and
dendritic morphology can persist in the absence of further stimulation.
We have used female sexual behavior in Syrian hamsters as a model of experience-based
plasticity in the brain (Hedges et al., 2010, Meisel & Mullins, 2006). This is a valuable
model because repeated sexual experience reliably produces changes analogous to chronic
exposure to drugs of abuse (Bradley et al., 2005a, Bradley & Meisel, 2001, Kohlert &
Meisel, 1999, Meisel & Joppa, 1994, Meisel et al., 1996, Meisel & Mullins, 2006).
Furthermore, previous work in our laboratory has demonstrated that overexpression of
ΔFosB in NAc using adeno-associated viral (AAV) vectors mimics the ability of sexual
experience to cause the formation of a conditioned place preference (Hedges et al., 2009),
suggesting that ΔFosB accumulation in NAc is sufficient to create the behavioral plasticity
associated with sexual experience. It remains unknown, however, whether ΔFosB is also
necessary for these behavioral consequences of sexual experience. The current study
therefore used AAV vectors to overexpress ΔJunD, a dominant negative inhibitor of ΔFosB,
in the NAc. We report here that overexpression of ΔJunD prevents the formation of a
conditioned place preference following repeated sexual experiences. When coupled with our
previous results, these results suggest that ΔFosB in NAc is both necessary and sufficient for
behavioral plasticity following sexual experience.
MATERIALS AND METHODS
Subjects—Adult female and male Syrian hamsters (Mesocricetus auratus) were purchased
from Charles River Laboratories (Wilmington, MA, USA) at approximately 60 days of age.
Females were used as experimental subjects, whereas males were used as sex stimulus
animals during conditioned place preference tests. Females were housed individually and
males were housed in pairs in polycarbonate cages (females: 50.8 × 40.6 × 20.3 cm; males:
43.2 × 22.9 × 20.3 cm). All animals were maintained on a reversed 14 hr light/10 hour dark
photoperiod (lights off between 1:00 and 11:00 PM) and all behavioral testing occurred
during the dark phase. The animal room was maintained at a controlled temperature of 22°C
and food and water were available ad libitum. All animal procedures were carried out in
accordance with the National Institutes of Health Guide for the Care and Use of Laboratory
Animals (NIH Publications No. 80-23; revised 1996) and approved by the University of
Minnesota Institutional Animal Care and Use Committee.
Viral Vectors—AAV is characterized by its ability to efficiently transfect neurons and
maintain specific transgene expression for long periods of time (Chamberlin et al., 1998).
AAV vectors exist in different serotypes based on the characterization of their capsid protein
coat. This experiment used an AAV2 (serotype 2) from Stratagene with a titer of over 108/μl
expressing green fluorescent protein (AAV-GFP) as well as an AAV vector that contained
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constructs for both GFP and ΔJunD (AAV-GFP-ΔJunD). ΔJunD decreases ΔFosB-mediated
transcription by competitively heterodimerizing with ΔFosB before binding the AP-1 region
within gene promoters. These AAV vectors mediate transgene expression in rats and mice
that becomes maximal within 10 days of injection and then persists at this level for at least
six months (Winstanley et al., 2007, Zachariou et al., 2006). Importantly, the vectors infect
neurons only and produce no toxicity greater than vehicle infusions alone. Details for the
production and use of these vectors are provided in earlier publications (Winstanley et al.,
2007, Zachariou et al., 2006).
Surgical Procedures
Ovariectomy—Female hamsters were bilaterally ovariectomized under sodium
pentobarbital anesthesia (Nembutal; 8.5 mg per 100 g body weight, i.p.). Subjects’ ovaries
were removed through bilateral flank incisions via cauterization of the uterine horn and
blood vessels. Chromic gut absorbable suture (Henry Schein, Melville, NY, USA) and
wound clips were used to close the smooth muscle and skin incisions, respectively.
Viral Vector Delivery—Immediately following ovariectomy, females were given
supplemental anesthesia if necessary, and then underwent stereotaxic delivery of either
AAV-GFP or AAV-GFP-ΔJunD. Anesthetized subjects were secured in the stereotaxic
apparatus such that their skull was level in the anterior-posterior (A-P) and medial-lateral
(M-L) planes. Following a midline scalp incision, the skin and temporal muscles were
retracted to expose the skull and a hand-operated drill was used to expose dura. All A-P and
M-L measurements were taken in mm relative to bregma and all dorsal-ventral (D-V)
measurements were taken in mm relative to dura. Bilateral stereotaxic injections were made
by lowering a microinjection syringe (model number, Hamilton, Reno, NV, USA) under
stereotaxic control (Microinjection Unit, Model 5002, David Kopf Instruments, Tujunga,
CA, USA) into bilateral sites targeting NAc and injecting 0.7 μl of AAV. To minimize the
flow of AAV up the needle tract, the syringe was left in place for 10 min after each
injection. Viral vectors were injected into NAc at least 2 weeks prior to behavior testing to
allow ΔJunD overexpression to develop.
Behavioral Testing
Sexual Experience—Ovariectomized hamsters were hormone-primed for weekly sexual
experiences via subcutaneous injections of estradiol benzoate (10 μg in 0.1 ml of cottonseed
oil) at approximately 48 and 24 hr prior to the sexual behavior test followed by a
subcutaneous injection of progesterone (500 μg in 0.1 ml of cottonseed oil) 4–6 hr prior to
the sexual behavior test. Females that received sexual experience were paired with a
sexually-experienced male hamster for a 10 minute session. Measures of female copulatory
behavior (lordosis latency and total lordosis duration) were scored live during each sexual
experience. Each male and female was only paired once.
Conditioned Place Preference—Our conditioned place preference (CPP) apparatus
consists of two lateral chambers (60 × 45 × 38 cm) connected by a clear central chamber (37
× 22 × 38 cm). The lateral chambers are differentiated by the color of the walls and the type
of bedding on the chamber floor: one lateral chamber is gray and contains aspen bedding
(Harlan Laboratories, IN, USA) while the other lateral chamber is white and contains
corncob bedding (Harlan Laboratories, IN, USA). Female hamsters underwent three
different types of behavioral tests in the CPP apparatus: a pre-test, 5 conditioning tests, and a
post-test. During the pre-test, hormone-primed female hamsters were placed in the clear
central chamber and allowed to freely explore the entire apparatus for 10 min in order to
establish an initial preference for one of the lateral compartments. During conditioning tests,
hormone-primed females were given sexual experience (see above) in their non-preferred
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chamber for 10 min. One hour after sexual conditioning in the non-preferred chamber,
females were placed into the initially preferred chamber alone for 10 min. The order in
which the conditioning stimuli (i.e., sexually-experienced male versus no stimulus) are
presented does not impact the formation or strength of a subsequent conditioned place
preference (Meisel & Joppa, 1994). Consequently, we chose a single order of presentation in
which subjects were always given sexual experience in the non-preferred chamber before
being placed alone in the preferred chamber during conditioning tests. After 5 consecutive
weeks of conditioning tests, hormone-primed females were given a post-test during which
they were again allowed to freely explore the entire apparatus to determine if their initial
preference had changed. Previous research has demonstrated that five consecutive sexual
experiences are sufficient to detect significant changes in place preference (Hedges et al.,
2009, Meisel & Joppa, 1994, Meisel et al., 1996). For both AAV-GFP and AAV-GFP-
ΔJunD groups, some animals did not receive sexual experience during the conditioning tests
but were hormone-primed and placed alone in each chamber for 10 min. As such, there were
four experimental groups in this experiment: A positive control group that received bilateral
AAV-GFP injections and were given sexual experience during conditioning (n = 7); a
negative control group that received bilateral AAV-GFP injections but were not given
sexual experience during conditioning (n = 7); a second negative control group that received
bilateral AAV-GFP-ΔJunD injections but were not given sexual experience during
conditioning (n = 6); and an experimental group that received bilateral AAV-GFP-ΔJunD
injections and were given sexual experience during conditioning (n = 8). All tests in the CPP
apparatus were conducted and scored by an observer blind to the experimental group of the
subjects.
Histology and Injection Verification
Following the last behavioral test, subjects were injected with an overdose of Sleepaway
(0.2 ml i.p., Fort Dodge Laboratories, Fort Dodge, IA, USA), injected intracardially with 0.2
ml of heparin sulfate (1000 IU/ml, Sagent Pharmaceuticals, Schaumburg, IL, USA) and
intracardially perfused with 25 mM PBS for 2 min (approximately 50 ml) followed by 4%
paraformaldehyde in 25 mM PBS for 20 min (approximately 500 ml). The brains were
removed and post-fixed for 1 hr in 4% paraformaldehyde then placed in a 10% sucrose
solution in PBS overnight. Serial coronal sections (40-μm) of frozen brain tissue were
sectioned on a microtome and every third section was processed for immunohistochemical
localization of either GFP or JunD. Free-floating sections were rinsed in PBS with 0.1%
bovine serum albumin (wash buffer) and then incubated in primary antibody (rabbit anti-
GFP: 1:1,000, AB3080, Millipore, Billerca, MA, USA; rabbit anti-JunD: 1:5000, sc-74,
Santa Cruz Biotechnology, Dallas, TX, USA) in wash buffer with 0.3% Triton-X at room
temperature for 24 hr. After rinsing in wash buffer, sections were incubated in a biotinylated
secondary antibody (1:200, Vector Laboratories, Burlingame, CA, USA) for 45 min at room
temperature, rinsed in wash buffer, and then incubated in an avidin-biotin complex
(Vectastain ABC Kit, Vector Laboratories) for 45 min at room temperature. Sections were
then rinsed in wash buffer and reacted in a 3, 3′-diaminobenzidine (DAB) solution with
0.003% hydrogen peroxide. After 5 min, sections were rinsed in wash buffer to stop the
chromagen reaction. Immunostained sections were mounted onto glass slides, coverslipped,
and examined under a light microscope for the location and rostral-caudal spread of AAV
injection as compared with published hamster neuroanatomical plates (Morin & Wood,
2001).
Statistical analysis
Female copulatory parameters during sexual conditioning tests were compared between
AAV conditions (AAV-GFP-ΔJunD vs. AAV-GFP) using one-way repeated measures
ANOVAs. Our operational definition for sexual reward was a significant increase in time
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spent in the conditioned chamber on the post-test compared to the pre-test. To test our
hypothesis that ΔFosB in the nucleus accumbens is necessary for sexual reward, conditioned
place preference data from each treatment group were analyzed individually with a repeated
measures t-test comparing the amount of time spent in the initially non-preferred chamber
between the pre-test and the post-test. Bonferroni corrections were applied to minimize the
likelihood of Type I error.
RESULTS
Immunohistochemical Characterization of AAV Injections
Brain tissue processed for immunohistochemical localization of either GFP or JunD was
examined under a light microscope to determine the placement and extent of AAV
injections. Injection placement was determined by tracing residual needle tracks. Bilateral
AAV injections were consistently located in the dorsal NAc core (Figure 1A). Of the 29
animals analyzed, 12 animals had injection sites in the mid-caudal NAc core (Bregma + 2.1
mm), 9 animals had had injection sites located slightly more caudally (Bregma + 1.8 mm),
and 8 animals had injection sites located slightly more rostrally (Bregma + 2.4 mm).
The extent of AAV infection was determined by examining JunD (Figure 1B) or GFP
(Figure 1C) expression. All animals injected with either AAV-GFP (n = 15) or AAV-GFP-
ΔJunD (n = 14) had significant transgene expression around the injection site that extended
within the NAc core in both the rostral-caudal and dorsal-ventral planes. In most animals (n
= 21), expression also extended into the NAc shell, albeit often to a lesser extent. In 6
animals, expression extended into the most rostral aspects of BNST, but labeling here was
sparse. In one animal, the injection site was visible within the dorsal NAc core, but infection
did not spread significantly outside of the region of the needle tip; as such, this animal was
removed from the analysis. Although the exact shape of the spread of infection varied
among individual animals, this variability was consistent between experimental groups.
Conditioned Place Preference
During the pre-test, 21 females demonstrated an initial preference for the white chamber and
8 females initially preferred the gray chamber. For those animals who received sexual
experience, there was no difference in the average lordosis latency (Figure 2A) or lordosis
duration (Figure 2B) between females injected with AAV-GFP and females injected with
AAV-GFP-ΔJunD on any test day (all p > 0.05), suggesting that the overexpression of GFP
or ΔJunD alone does not affect the receptive behavior of females.
For each group, the amount of time spent in the initially non-preferred chamber during the
pre-test was compared to the time spent in the same chamber during the post-test (Figure 3).
If sexual conditioning was successful, then the amount of time spent in the initially non-
preferred chamber should increase significantly following sexual experience. Indeed, the
positive control group that received AAV-GFP injections and was given sexual experience
spent significantly more time in the initially non-preferred chamber during the post-test
compared to during the pre-test (t(6)= 8.21, P < 0.05). In contrast, the amount of time spent
in the initially non-preferred chamber did not differ between the pre- and post-tests for
AAV-GFP-injected animals who did not receive sexual experience (t(6)= 0.58, P > 0.05).
Similarly, the amount of time spent in the initially non-preferred chamber did not differ
between the pre- and post-tests for AAV-GFP-ΔJunD-injected animals who did not receive
sexual experience (t(5)= 0.92, P > 0.05). Interestingly, females in the AAV-GFP-ΔJunD
group who received sexual experience also did not demonstrate a significant increase in the
amount of time spent in the initially non-preferred chamber during the post-test (t(7)= 1.01,
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P > 0.05), suggesting that the overexpression of ΔJunD in NAc can prevent the formation of
a conditioned place preference as a result of sexual experience.
DISCUSSION
The hypothesized role of ΔFosB in mediating the neural and behavioral responses to natural
rewards and drugs of abuse is based mainly on studies demonstrating that repeated
experience with these stimuli correlates with the accumulation ΔFosB in the NAc (Meisel &
Mullins, 2006, Pitchers et al., 2010c, Teegarden & Bale, 2007, Wallace et al., 2008, Werme
et al., 2002, Zachariou et al., 2006) or that supraphysiological expression of ΔFosB in the
NAc can augment the motivation for or response to these stimuli (Colby et al., 2003, Hedges
et al., 2009, Pitchers et al., 2010c, Pitchers et al., 2013, Zachariou et al., 2006). Fewer
studies, however, have attempted to experimentally manipulate the endogenous activity of
ΔFosB in the brain to directly test the role of ΔFosB in reinforcement and reward. Our study
therefore contributes to an important and growing body of literature demonstrating the
necessity of endogenous ΔFosB expression in the NAc for adaptive responding to rewarding
stimuli (Olausson et al., 2006, Peakman et al., 2003, Pitchers et al., 2010c, Pitchers et al.,
2013, Werme et al., 2002).
In the current study, overexpression of ΔJunD was targeted to the NAc core and resulted in
minimal spread to the NAc shell or other brain areas outside of the NAc. The ability of core-
centered injections of ΔJunD to mitigate the rewarding consequences of sexual experience as
measured in a conditioned place preference task is in agreement with previous research in
our laboratory demonstrating that sexual experience increases c-Fos (Bradley & Meisel,
2001) and FosB expression (Meisel & Mullins, 2006) specifically within the NAc core. In
addition, sexual experience (Meisel & Mullins, 2006) and another naturally rewarding
behavior, aggressive experience (Staffend & Meisel, 2012), both increase dendritic spine
density exclusively within the NAc core. Furthermore, the neural circuitry mediating the
behavioral response to natural (e.g., sexual behavior) or synthetic (e.g., experience with
drugs of abuse) rewards likely overlaps substantially, and recent evidence suggests that the
NAc core may be particularly important for conditioning to social stimuli. In male rats, the
acquisition of CPP for both social interaction and cocaine increases expression of the
immediate early gene, Zif268, in the NAc shell and core (El Rawas et al., 2012). In the NAc
core, however, CPP for social interaction increased Zif268 expression relative to CPP for
cocaine, suggesting that the NAc core may be preferentially involved in the conditioning to
naturally rewarding stimuli (El Rawas et al., 2012). It is also possible that separate
populations of cells within both the shell and core may respond differentially to naturally
rewarding stimuli versus drug stimuli, as neurons in both subregions of the NAc show
similar neuronal activity during operant responding to two natural reinforcers (i.e., food and
water), but different firing patterns during responding for a natural reinforcer versus cocaine
(Carelli et al., 2000).
As in female hamsters, sexual experience in male rats produces long-lasting neural and
behavioral changes that appear to depend on ΔFosB signaling in the NAc. Sexual experience
in male rats results in the accumulation of FosB protein in the NAc (Pitchers et al., 2010c).
Furthermore, viral overexpression of ΔFosB in the NAc facilitates copulatory efficiency in
males, whereas viral overexpression of ΔJunD in the NAc inhibits experience-induced
facilitation of male sexual behavior (Pitchers et al., 2010c, Pitchers et al., 2013). This ability
of local manipulation of ΔFosB levels in the NAc to affect the overt expression of sexual
behavior appears to be unique to males. Whereas overexpression of ΔJunD increases males’
latency to mount, intromit, and ejaculate during sexual experience sessions (Pitchers et al.,
2013), overexpression of either ΔFosB in a previous study (Hedges et al., 2009) or ΔJunD in
the current study affected the rewarding consequences of sexual behavior without affecting
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lordosis latency or duration in female hamsters. As our belief is that nucleus accumbens
ΔFosB is a mediator of sexual motivation in both males and females, the apparent difference
in the effects of ΔJunD on copulation in males and females may lie more in the fact that
copulatory parameters in males includes both motivational and motoric components,
whereas the lordosis response in females is primarily a “reflexive” behavior uncoupled from
sexual motivation (Rivas & Mir, 1991).
Although the mechanisms underlying sexual experience-induced changes in ΔFosB
signaling have not been completely defined, research in our laboratory and others suggests
that elevations in NAc dopamine (DA) may drive this plasticity via D1 receptor activation.
In female hamsters, sexual experience increases extracellular DA in the NAc (Kohlert &
Meisel, 1999) and 6-hydroxydopamine lesions of the NAc block the experience-induced
increase in female copulatory efficiency in future tests with males (Bradley et al., 2005b).
Our assumption is that these effects are mediated by dopamine D1 neurotransmission, as
sexual experience increases D1 receptor-mediated cyclic AMP production in the NAc
compared with the response in sexually naïve female hamsters (Bradley et al., 2004).
Further, our recent work demonstrates that sexual experience increases dendritic spine
density specifically on D1-bearing medium spiny neurons in the NAc core of female
hamsters (Staffend and Meisel, in preparation). Complementary evidence from male rats has
demonstrated that sexual experience-induced increases in ΔFosB accumulation are
dependent on mating-induced D1 receptor activation in the NAc as well. For example,
ΔJunD overexpression blocks the sexual experience-induced increase in dendritic spine
density in the NAc and pharmacological blockage of D1 receptors during sexual behavior
prevents increases in dendritic spine density in males (Pitchers et al., 2013). Our working
hypothesis is that sexual experience drives behavioral and structural plasticity through
dopamine D1-mediated activation of signaling pathways that induce ΔFosB expression.
The model of experienced-based changes in ΔFosB signaling depending on D1 activation in
the NAc is not limited to naturally rewarding experiences such as sexual behavior. Recent
research by Grueter et al. (2013) has demonstrated that overexpression of ΔFosB in the NAc
changes the synaptic properties of D1-expressing neurons in the NAc and increases dendritic
spine density specifically on D1-bearing neurons in the NAc. Furthermore, selective
overexpression of ΔFosB in D1 but not D2 neurons enhances locomotor sensitization to
cocaine as well as CPP for cocaine (Grueter et al., 2013). Given the shared neural substrates
for responding to natural rewards and drugs of abuse, and the knowledge that sexual
experience can sensitize behavioral responses to drugs of abuse (Bradley & Meisel, 2001,
Pitchers et al., 2010a, Pitchers et al., 2013), it is important to understand how experience
with natural rewards can impact the vulnerability to drug addiction. By comparing the neural
changes that result from naturally rewarding behaviors to those seen after exposure to drugs
of abuse, we can separate the neural properties of drug addiction from the endogenous
plasticity that underlie activities in everyday life.
Acknowledgments
The authors wish to thank Calyn Maske for her assistance collecting and scoring behavioral data. Research reported
in this publication was supported by DA013680 to RLM and MH51399 to EJN. In addition, LEB was supported by
The National Institutes on Drug Abuse under award number T32DA007234. The content is solely the responsibility
of the authors and does not necessarily represent the official views of the National Institutes of Health.
References
Bradley KC, Boulware MB, Jiang H, Doerge RW, Meisel RL, Mermelstein PG. Changes in gene
expression within the nucleus accumbens and striatum following sexual experience. Genes Brain
Behav. 2005a; 4:31–44. [PubMed: 15660666]
Been et al. Page 7
Genes Brain Behav. Author manuscript; available in PMC 2014 August 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Bradley KC, Haas AR, Meisel RL. 6-Hydroxydopamine lesions in female hamsters (Mesocricetus
auratus) abolish the sensitized effects of sexual experience on copulatory interactions with males.
Behav Neurosci. 2005b; 119:224–232. [PubMed: 15727527]
Bradley KC, Meisel RL. Sexual behavior induction of c-Fos in the nucleus accumbens and
amphetamine-stimulated locomotor activity are sensitized by previous sexual experience in female
Syrian hamsters. J Neurosci. 2001; 21:2123–2130. [PubMed: 11245696]
Bradley KC, Mullins AJ, Meisel RL, Watts VJ. Sexual experience alters D1 receptor-mediated cyclic
AMP production in the nucleus accumbens of female Syrian hamsters. Synapse. 2004; 53:20–27.
[PubMed: 15150737]
Carelli RM, Ijames SG, Crumling AJ. Evidence that separate neural circuits in the nucleus accumbens
encode cocaine versus “natural” (water and food) reward. J Neurosci. 2000; 20:4255–4266.
[PubMed: 10818162]
Chamberlin NL, Du B, de Lacalle S, Saper CB. Recombinant adeno-associated virus vector: use for
transgene expression and anterograde tract tracing in the CNS. Brain Res. 1998; 793:169–175.
[PubMed: 9630611]
Chen BT, Hopf FW, Bonci A. Synaptic plasticity in the mesolimbic system: therapeutic implications
for substance abuse. Ann N Y Acad Sci. 2010; 1187:129–139. [PubMed: 20201850]
Chen J, Kelz MB, Hope BT, Nakabeppu Y, Nestler EJ. Chronic Fos-related antigens: stable variants of
deltaFosB induced in brain by chronic treatments. J Neurosci. 1997; 17:4933–4941. [PubMed:
9185531]
Colby CR, Whisler K, Steffen C, Nestler EJ, Self DW. Striatal cell type-specific overexpression of
DeltaFosB enhances incentive for cocaine. J Neurosci. 2003; 23:2488–2493. [PubMed: 12657709]
Couppis MH, Kennedy CH. The rewarding effect of aggression is reduced by nucleus accumbens
dopamine receptor antagonism in mice. Psychopharmacology. 2008; 197:449–456. [PubMed:
18193405]
El Rawas R, Klement S, Kummer KK, Fritz M, Dechant G, Saria A, Zernig G. Brain regions
associated with the acquisition of conditioned place preference for cocaine vs. social interaction.
Front Behav Neurosci. 2012; 6:63. [PubMed: 23015784]
Greenwood BN, Foley TE, Le TV, Strong PV, Loughridge AB, Day HE, Fleshner M. Long-term
voluntary wheel running is rewarding and produces plasticity in the mesolimbic reward pathway.
Behav Brain Res. 2011; 217:354–362. [PubMed: 21070820]
Grueter BA, Robison AJ, Neve RL, Nestler EJ, Malenka RC. ΔFosB differentially modulates nucleus
accumbens direct and indirect pathway function. Proc Nat Acad Sci. 2013; 110:1923–1928.
[PubMed: 23319622]
Hedges VL, Chakravarty S, Nestler EJ, Meisel RL. Delta FosB overexpression in the nucleus
accumbens enhances sexual reward in female Syrian hamsters. Genes Brain Behav. 2009; 8:442–
449. [PubMed: 19566711]
Hedges VL, Staffend NA, Meisel RL. Neural mechanisms of reproduction in females as a predisposing
factor for drug addiction. Front Neuroendocrinol. 2010; 31:217–231. [PubMed: 20176045]
Kohlert JG, Meisel RL. Sexual experience sensitizes mating-related nucleus accumbens dopamine
responses of female Syrian hamsters. Behav Brain Res. 1999; 99:45–52. [PubMed: 10512571]
Meisel RL, Joppa MA. Conditioned place preference in female hamsters following aggressive or
sexual encounters. Physiol Behav. 1994; 56:1115–1118. [PubMed: 7824580]
Meisel RL, Joppa MA, Rowe RK. Dopamine receptor antagonists attenuate conditioned place
preference following sexual behavior in female Syrian hamsters. Eur J Pharm. 1996; 309:21–24.
Meisel RL, Mullins AJ. Sexual experience in female rodents: cellular mechanisms and functional
consequences. Brain Res. 2006; 1126:56–65. [PubMed: 16978593]
Morin, LP.; Wood, RI. A Stereotaxic Atlas of the Golden Hamster Brain. Academic Press; San Diego:
2001.
Nestler EJ. Review. Transcriptional mechanisms of addiction: role of DeltaFosB. Philos Trans R Soc
Lond B Biol Sci. 2008; 363:3245–3255. [PubMed: 18640924]
Olausson P, Jentsch JD, Tronson N, Neve RL, Nestler EJ, Taylor JR. DeltaFosB in the nucleus
accumbens regulates food-reinforced instrumental behavior and motivation. J Neurosci. 2006;
26:9196–9204. [PubMed: 16957076]
Been et al. Page 8
Genes Brain Behav. Author manuscript; available in PMC 2014 August 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Peakman MC, Colby C, Perrotti LI, Tekumalla P, Carle T, Ulery P, Chao J, Duman C, Steffen C,
Monteggia L, Allen MR, Stock JL, Duman RS, McNeish JD, Barrot M, Self DW, Nestler EJ,
Schaeffer E. Inducible, brain region-specific expression of a dominant negative mutant of c-Jun in
transgenic mice decreases sensitivity to cocaine. Brain Res. 2003; 970:73–86. [PubMed:
12706249]
Pitchers KK, Balfour ME, Lehman MN, Richtand NM, Yu L, Coolen LM. Neuroplasticity in the
mesolimbic system induced by natural reward and subsequent reward abstinence. Biol Psychiatry.
2010a; 67:872–879. [PubMed: 20015481]
Pitchers KK, Balfour ME, Lehman MN, Richtand NM, Yu L, Coolen LM. Neuroplasticity in the
mesolimbic system induced by natural reward and subsequent reward abstinence. Biol Psychiatry.
2010b; 67:872–879. [PubMed: 20015481]
Pitchers KK, Frohmader KS, Vialou V, Mouzon E, Nestler EJ, Lehman MN, Coolen LM. DeltaFosB
in the nucleus accumbens is critical for reinforcing effects of sexual reward. Genes Brain Behav.
2010c; 9:831–840. [PubMed: 20618447]
Pitchers KK, Vialou V, Nestler EJ, Laviolette SR, Lehman MN, Coolen LM. Natural and Drug
Rewards Act on Common Neural Plasticity Mechanisms with DeltaFosB as a Key Mediator. J
Neurosci. 2013; 33:3434–3442. [PubMed: 23426671]
Rivas FJ, Mir D. Accumbens lesion in female rats increases mount rejection without modifying
lordosis. Rev Esp Fisiol. 1991; 47:1–6. [PubMed: 1871414]
Robinson TE, Kolb B. Structural plasticity associated with exposure to drugs of abuse.
Neuropharmacology. 2004; 47(Suppl 1):33–46. [PubMed: 15464124]
Robison AJ, Nestler EJ. Transcriptional and epigenetic mechanisms of addiction. Nat Rev Neurosci.
2011; 12:623–637. [PubMed: 21989194]
Russo SJ, Dietz DM, Dumitriu D, Morrison JH, Malenka RC, Nestler EJ. The addicted synapse:
mechanisms of synaptic and structural plasticity in nucleus accumbens. Trends Neurosci. 2010;
33:267–276. [PubMed: 20207024]
Staffend NA, Meisel RL. Aggressive experience increases dendritic spine density within the nucleus
accumbens core in female Syrian hamsters. Neuroscience. 2012; 227:163–169. [PubMed:
23041760]
Teegarden SL, Bale TL. Decreases in dietary preference produce increased emotionality and risk for
dietary relapse. Biol Psychiatry. 2007; 61:1021–1029. [PubMed: 17207778]
Vargas-Perez H, Mena-Segovia J, Giordano M, Diaz JL. Induction of c-fos in nucleus accumbens in
naive male Balb/c mice after wheel running. Neurosci Lett. 2003; 352:81–84. [PubMed:
14625028]
Wallace DL, Vialou V, Rios L, Carle-Florence TL, Chakravarty S, Kumar A, Graham DL, Green TA,
Kirk A, Iniguez SD, Perrotti LI, Barrot M, DiLeone RJ, Nestler EJ, Bolanos-Guzman CA. The
influence of DeltaFosB in the nucleus accumbens on natural reward-related behavior. J Neurosci.
2008; 28:10272–10277. [PubMed: 18842886]
Werme M, Messer C, Olson L, Gilden L, Thoren P, Nestler EJ, Brene S. Delta FosB regulates wheel
running. J Neurosci. 2002; 22:8133–8138. [PubMed: 12223567]
Winstanley CA, LaPlant Q, Theobald DE, Green TA, Bachtell RK, Perrotti LI, DiLeone RJ, Russo SJ,
Garth WJ, Self DW, Nestler EJ. DeltaFosB induction in orbitofrontal cortex mediates tolerance to
cocaine-induced cognitive dysfunction. J Neurosci. 2007; 27:10497–10507. [PubMed: 17898221]
Zachariou V, Bolanos CA, Selley DE, Theobald D, Cassidy MP, Kelz MB, Shaw-Lutchman T, Berton
O, Sim-Selley LJ, Dileone RJ, Kumar A, Nestler EJ. An essential role for DeltaFosB in the
nucleus accumbens in morphine action. Nat Neurosci. 2006; 9:205–211. [PubMed: 16415864]
Been et al. Page 9
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Figure 1. AAV injections
A) AAV injections for the four experimental groups were localized to the dorsal nucleus
accumbens core. Circles represent animals injected with AAV-GFP-ΔJunD, whereas squares
represent animals injected with AAV-GFP. Filled symbols represent animals that received
sexual experience, whereas open symbols represent animals that remained sexually-naïve.
Numbers represent distance relative to bregma, ac, anterior commissure. B)
Photomicrograph of representative nuclear immunostaining for JunD protein, scale bar =
200 μm. C) Photomicrograph of representative immunostaining for GFP in cell bodies and
processes.
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Figure 2. Overexpression of ΔJunD in the NAc does not affect the expression of female sexual
behavior
During sexual experience trials, females injected with AAV-GFP-ΔJunD did not differ from
females injected with AAV-GFP in A) the latency to express lordosis or B) the total duration
of lordosis on any test day. Data expressed as means ± standard errors.
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Figure 3. Overexpression of ΔJunD in the NAc prevents the formation of conditioned place
preference following sexual experience
During the post-test, females who were injected with AAV-GFP and received sexual
experience spent significantly more time in their initially non-preferred chamber (i.e., the
chamber in which they received sexual experience) than they did during the pre-test. In
contrast, the amount of time spent in the non-preferred chamber did not differ between the
pre- and post-test for females injected with AAV-GFP-ΔJunD that received sexual
experience. Likewise, females who were injected with either AAV-GFP or AAV-GFP-
ΔJunD and did not receive sexual experience did not differ in the amount of time they spent
in the non-preferred chamber during the pre- and post-tests. Data expressed as means ±
standard errors. *significant difference between pre- and post-test (p < 0.05)
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