Activation of GSK3\xce\xb2 induced by recall of cocaine reward\nmemories is dependent on GluN2A/B NMDA receptor signaling
Glycogen synthase kinase-3\xce\xb2 (GSK3\xce\xb2) is a critical regulator of the balance between long-term depression and long-term potentiation which is essential for learning and memory. Our previous study demonstrated that GSK3\xce\xb2 activity is highly induced during cocaine memory reactivation, and that reconsolidation of cocaine reward memory is attenuated by inhibition of GSK3\xce\xb2. NMDA receptors and protein phosphatase 1 (PP1) are activators of GSK3\xce\xb2. Thus, this study investigated the roles of NMDA receptor subtypes and PP1 in the reconsolidation of cocaine contextual reward memory. Cocaine contextual memories were established and evaluated using cocaine conditioned place preference methods. The regulation of GSK3\xce\xb2 activity in specific brain areas was assessed by measuring its phosphorylation state using immunoblot assays. Mice underwent cocaine place conditioning for 8 days and were tested for place preference on day 9. Twenty-four hours later, mice were briefly confined to the compartment previous paired with cocaine to reactivate cocaine-associated memories. Administration of the GluN2A- and GluN2B-NMDA receptor antagonists, NVP-AAM077 and ifenprodil respectively, immediately following recall abrogated an established cocaine place preference, while preventing the activation of GSK3\xce\xb2 in the amygdala, nucleus accumbens, and hippocampus during cocaine memory reactivation. PP1 inhibition with okadaic acid also blocked the activation of GSK3\xce\xb2 and attenuated a previously established cocaine place preference. These findings suggest that the dephosphorylation of GSK3\xce\xb2 that occurred upon activation of cocaine-associated reward memories may be initiated by the activation of PP1 during the induction of NMDA receptor-dependent reconsolidation of cocaine mnemonic traces. Moreover, the importance of NMDA receptors and PP1 in reconsolidation of cocaine memory makes them potential therapeutic targets in treatment of cocaine use disorder and prevention of relapse.
Introduction<!>Animals<!>Drugs<!>Cocaine Conditioned Place Preference<!>Protein Measurements by Immunoblotting<!>Intracranial implantation of injection cannulas<!>Experiment 1: Effect of the GluN2A antagonist, NVP-AAM077, on the\nreconsolidation of cocaine reward memory.<!>Experiment 2: Effect of the NMDAR GluN2B-selective antagonist,\nifenprodil, on the reconsolidation of cocaine reward memory.<!>Experiment 3: Effect of the NMDAR GluN2A-preferring antagonist on the\ndephosphorylation of GSK3\xce\xb2 induced by reactivation of cocaine reward\nmemory.<!>Experiment 4: Effect of the NMDAR GluN2B-selective antagonist ifenprodil\non the dephosphorylation of GSK3\xce\xb2 induced by reactivation of cocaine\nreward memory.<!>Experiment 5: Effect of PP1 inhibition on GSK3\xce\xb2 phosphorylation\nduring memory reactivation.<!>Experiment 6: Effect of the PP1 inhibitor, okadaic acid, on the\nreconsolidation of cocaine reward memory.<!>Statistical Analysis<!>Experiment 1: The GluN2A-preferring antagonist NVP-AAM077 disrupted an\nestablished cocaine place preference<!>Experiment 2: The selective GluN2B antagonist ifenprodil erased a previously\nestablished cocaine place preference<!>Experiment 3: Blockade of GluN2A-containing NMDA receptors prevented\nactivation of GSK3\xce\xb2 by cocaine memory recall<!>Experiment 4: Blockade of GluN2B-containing NMDA receptors prevented\nactivation of GSK3\xce\xb2 produced by cocaine memory recall<!>Experiment 5: Inhibition of PP1 blocked the activation of GSK3\xce\xb2\nproduced by re-exposure to the cocaine-paired environment.<!>Experiment 6: Inhibition of protein phosphatase 1 abrogated a previously\nestablished cocaine-place preference.<!>Discussion<!>Conclusions
<p>Craving elicited by exposure to environmental cues that were previously
associated with use of addictive drugs is a primary contributor to relapse (O'Brien et al. 1990).
Memories of drug-paired cues are highly engrained and difficult to erase in both
humans (Ehrman et al. 1992;
Gawin 1991) and experimental animals
(de Wit & Stewart 1981). After
reactivation, memories undergo a process of reconsolidation which strengthens the
memory trace. However, the reactivation period is a critical time when memory traces
become labile and can be manipulated behaviorally or pharmacologically by
interfering with reconsolidation (Nader et
al. 2000). Thus, targeting the reconsolidation process
related to the maintenance of drug memory has therapeutic potential for the
treatment of addiction and the prevention of cue-induced relapse.</p><p>NMDA receptors play a crucial role in reconsolidation of cocaine-related
memory (Alaghband & Marshall 2013; Itzhak 2008; Milton et al. 2008), likely through bidirectional
effects on synaptic plasticity, LTP and LTD (Sajikumar & Frey 2004). NMDARs are composed of GluN1 subunits, GluN2
subunits (A,B,C,D), and GluN3 subunits (Cull-Candy
& Leszkiewicz 2004; Paoletti
2011; Traynelis et al.
2010). In the adult, GluN2A and GluN2B are the predominant subunits in
the hippocampus, amygdala, striatum and cortex (Lopez de Armentia & Sah 2003; Monyer
et al. 1992; Wenzel
et al. 1995) and play a central role in synaptic
function and plasticity. The subtypes of NMDA receptors that contribute to the
maintenance of conditioned fear memories have been investigated. Administration of
the GluN2B-selective NMDAR antagonist ifenprodil into the basolateral amygdala prior
to fear memory reactivation prevents the destabilization of the conditioned-fear
memory (Ben Mamou et al.
2006; Milton et al.
2013), whereas administration of the GluN2A-preferring NMDAR antagonist
NVP-AAM077 prior to the fear memory reactivation session reduces conditioned
freezing during subsequent testing (Milton et al,
2013). These findings suggest that GluN2B-NMDARs and GluN2A-NMDARs within
the basolateral amygdala are required for fear memory destabilization and
reconsolidation respectively (Milton et al,
2013). Although a pivotal role for NMDA receptors in the reconsolidation
of drug-cue memories is appreciated (Alaghband &
Marshall 2013; Brown et
al. 2008; Kelley et
al. 2007; Milton et
al. 2008; Sadler et
al. 2007; Wouda et
al. 2010), the subtype(s) of NMDA receptors involved is not
known and the signaling pathways remain under investigation.</p><p>Our previous study demonstrates that glycogen synthase kinase-3β
(GSK3β) activity is enhanced in the nucleus accumbens, hippocampus, and
prefrontal cortex during cocaine memory reactivation (Shi et al. 2014). Importantly,
pharmacological inhibition of GSK3β blocks reconsolidation of cocaine reward
memory and erases a previously established cocaine place preference indicating a
critical role of GSK3β signaling in the reconsolidation of cocaine-reward
memories (Shi et al. 2014).
GSK3β has a wide influence over neural function and plasticity due to its
ability to phosphorylate dozens of substrates including signaling proteins and
transcriptional factors (Jope & Roh 2006;
Kockeritz et al. 2006).
GSK3β may contribute to memory reconsolidation by controlling the balance
between long-term potentiation and LTD (Kimura
et al. 2008). NMDA receptors can activate
GSK3β through a process involving protein phosphatase 1 (PP1). PP1
dephosphorylates Ser9-GSK3β (Szatmari
et al. 2005), thereby increasing GSK3β
activity. In addition, PP1 indirectly activates GSK3β by dephosphorylating
Thr308-Akt, thus reducing Akt activity (Peineau
et al. 2008); Akt is a kinase that phosphorylates
GSK3β. Therefore, during LTD, activation of PP1 can activate GSK3β
both by direct dephosphorylation and indirectly through inhibition of Akt.</p><p>Our previous report proposed a model by which a protein phosphatase cascade,
such as PP2B and PP1, is activated down-stream of NMDA receptors during LTD,
resulting in the dephosphorylation of Akt and activation of GSK3β following
recall of cocaine contextual memories (Shi et al.
2014). The present study sought to test the validity of the model and
establish the mechanisms of regulation of GSK3β signaling that are involved
in the maintenance of cocaine memories. The contributions of specific NMDA receptor
subtypes and PP1 activity in the reconsolidation of cocaine contextual reward
memories were investigated.</p><!><p>Male CD-1 mice (Charles River Laboratories, Wilmington, MA. RRID:
SCR_003792), 8 weeks old on delivery (32–35g), were housed in group of 4
per cage (cage type: Mouse 750™ Ventilated Cage) without additional
enrichment objects, under a 12-hour light/dark cycle (7:00/19:00). Standard chow
and water were available ad libitum. Mice were housed for five
days prior to experimental procedures and were weighed daily. Behavioral tests
were conducted between 9:00–12:00 (Experiment 5–6), or
14:00–17:00 (Experiment 1–4). Mice were arbitrarily assigned to
experimental groups; no randomization was performed. Animal testing was
conducted in accordance with the National Institutes of Health guidelines for
the Care and Use of Laboratory Animals and with an approved protocol from Temple
University Institutional Animal Care and Use Committee (approval: ACUP 4600).
The experimental protocol was not pre-registered. No sample size calculation was
performed because the sample sizes were consistent with our previous reports
(Shi et al. 2014)
and those standard in the field. Six experiments were conducted in the present
study. A total of 249 mice entered into the cocaine conditioned place preference
procedure (described below). Three mice were removed from Experiment 6 because
the cannulas became dislodged (N=2) or were unhealthy (N=1). 246 mice were
tested for place preference on Day 9. Mice that did not have a place preference
score of >180 sec on Day 9 did not continue in the study; 66 mice
fulfilled this exclusion criteria and did not continue onto Day 10. 180 mice
continued onto the second phase of the study. 32 mice were used for saline
control experiments (Experiments 1 and 2) and received saline prior to being
placed into both compartments of the conditioning chambers. In this case, the
inclusion criteria was a place preference score of −359 sec to +359 sec.
24 mice fulfilled this criteria and continued to Day 10 of the study. Animal
procedures were performed by skilled and experienced experimenters to ensure
that no unnecessary pain or distress was caused as a result of the
procedures.</p><!><p>Cocaine hydrochloride was generously supplied by the National Institute
on Drug Abuse Drug Supply Program, dissolved in sterile saline, and injected
intraperitoneally (i.p.) in a volume of 3 ml/kg body weight. Okadaic acid (CAS#:
155751-72-7, 2014, 2019), NVP-AAM077 (CAS#:459836-30-7, 2014, 2017, 2018) and
ifenprodil (CAS#: 23210-58-4, 2014, 2017, 2018) were purchased from
Sigma-Aldrich (St. Louis, MO) and dissolved in sterile saline. An equal volume
of saline served as the vehicle control for all drugs.</p><!><p>Conditioned place preference chambers were rectangular in shape
(45×20×20cm) and consisted of two compartments, separated by a
removable door. One compartment had a smooth floor with white and black vertical
striped walls, whereas the other had a rough floor and white walls with black
circles. Illumination in both compartments was equal. An unbiased conditioned
place preference procedure was used, similar to that described in our previous
study (Shi et al, 2014). Place
conditioning occurred once daily for 8 days, wherein mice were injected with
saline or cocaine (10 mg/kg, i.p.) and placed into alternate compartments of the
conditioning chamber for 30 minutes, resulting in 4 pairings with saline and 4
pairings with cocaine. On Day 9, mice had access to both compartments for 30
minutes in a drug-free state and time in each compartment was recorded.
Preference scores were calculated as time in cocaine-paired minus time in
saline-paired compartments and reported in seconds. The investigators who
performed the daily place conditioning sessions were not blinded to
cocaine/saline administration. The experimenters who recorded the place
preference scores were blinded to the experimental groups. Mice that showed
cocaine place preference scores of >180 sec proceeded onto the
reconsolidation phase of the studies as described in Experiments 1–6
below.</p><!><p>The nucleus accumbens, hippocampus, and amygdala from individual mice
were sonicated in 100°C 1% sodium dodecyl sulfate with 1 mM NaF and 1 mM
Na3VO4 as phosphatase inhibitors, boiled for 5 minutes, and stored at
−80°C. Protein concentrations were determined by the Protein A280
method using a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific,
Waltham, MA, USA). Protein extracts (12.5 Î¼g) were separated on 7.5%
Tris–HCl Bio-Rad Ready-gels (Cat#: 4561026). Bio-Rad Laboratories,
Hercules, CA) and transferred onto nitrocellulose membranes. Membranes were
blocked in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) and
Tween-Tris-buffered saline, and incubated overnight at 4°C in the
following primary antibodies; anti-phospho-ser9-GSK3β (1:1000, Cat#9323,
RRID:AB_2115201, Cell Signaling, Beverly, MA) and anti-GSK3β (1:1000;
Cat#9315, RRID:AB_490890, Cell Signaling). Membranes were washed in
Tween-Tris-buffered saline and incubated with anti-rabbit (1:15,000, Cat#
926–32211, RRID:AB_621843) or anti-mouse (1:15, 000, Cat#
926–68070, RRID:AB_10956588) secondary antibodies conjugated to two
different infra-red dyes (LI-COR Biosciences, Lincoln, NE). Membranes were
visualized and proteins were quantified using the Odyssey infrared imaging
system and software (Li-COR). Phosphorylated and total forms of GSK3β
were detected simultaneously as the colors green and red respectively. Membranes
were stripped of antibodies using the New Blot nitro stripping buffer (LI-COR)
and re-probed with anti-β-tubulin (1:600,000; Cat#T8328, RRID:AB_1844090,
Sigma-Aldrich, St. Louis, MO). Ratios of densities of phosphorylated
GSK3β to total GSK3β; phospho-GSK3β to β-tubulin
levels, and total GSK3β to β-tubulin were calculated. The
experimenter performing the immunoblot assays was blind to animal treatment
groups.</p><!><p>For experiments requiring intracerebroventricular (i.c.v.) drug
infusions (Exp 5 and 6), indwelling cannula were implanted into the lateral
ventricle under general anesthesia. A mixture of ketamine HCl (100 mg/kg Rompum;
Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (20 mg/kg AnaSed;
Shenandoah, IA) was administered ip. This combination is considered to be a
first choice in rodents when injectable anesthetics are used because it provides
deep sedation, good immobilization with moderate analgesia (Flecknell et al. 1996). Anesthetized
mice were mounted in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA).
Stainless steel guide cannulas (PlasticsOne, Roanoke, VA, USA, 26 gauge) were
implanted into the lateral ventricle (unilateral) at the following coordinates
from bregma: AP:−0.5mm, ML:1.0mm, DV:2.5mm (Paxinos & Franklin 2001), cemented in place and
closed by stainless steel dummy cannulas of the same length (PlasticsOne,
C315DC). Meloxicam (1mg/kg, sc) was administered pre-and post-surgical
procedures for analgesia. Behavioral studies began 5–7 days after
surgery.</p><!><p>The aim of Experiments 1 and 2 was to determine which subtype(s) of
NMDA receptors contributes to the reconsolidation of cocaine-associated
memories. In Experiment 1, 40 mice underwent cocaine conditioned place
condition for 8 days and were tested for place preference on Day 9, as
described above. The preference scores of 29 mice fulfilled the inclusion
criteria and continued onto the second phase of the study. On Day 10, 24
hours following the test for cocaine place preference, 18 mice were
arbitrarily assigned to three groups (N=6 mice/group) and were re-exposed to
the compartment previously paired with cocaine for 10 min in order to
reactivate the cocaine-paired memories. Memory reactivation was followed
immediately by administration of the GluN2A-preferring antagonist NVP-AAM077
(0, 1 and 5 mg/kg, i.p.) (Fox et
al. 2006; Laird
et al. 2014). 11 mice were assigned to two
non-reactivation control groups (N=5–6 mice/group). As such, they
remained in their home cages and were injected with vehicle or NVP-AAM077 (5
mg/kg, i.p.) according to the same time schedule as the other three
experimental groups. On Days 11 and 18, all mice were re-tested for place
preference without further drug injections or conditioning sessions.</p><p>To test whether a single injection of NVP-AAM077 alone produces a
place preference or aversion, 16 mice were conditioned with saline in both
compartments for 8 days and tested on Day 9 for place preference. 12 mice
showed a place preference score between −359 sec and +359 sec and
continued onto the second phase of study. These mice were placed into the
chamber for 10 min and received either vehicle or 5 mg/kg of NVP-AAM077
immediately thereafter (N=6 mice/group). Mice were re-tested for place
preference without further drug injections or conditioning sessions on Days
11 and 18.</p><!><p>Experiment 2 was performed exactly as described in Experiment 1
except that mice were injected with the selective GluN2B antagonist
ifenprodil (0, 1, 5 and 10 mg/kg, i.p.) (Liddie & Itzhak 2016; Suzuki
et al. 1999) on Day 10 following re-exposure
to the cocaine-paired chamber or in the home-cage. 47 out of 64 mice showed
a cocaine place preference on Day 9 and were administered ifenprodil on Day
10 (N=6–9 mice/group). Likewise, the ability of a single injection of
ifenprodil (0, 5 mg/kg, i.p.) to produce a place aversion was tested by the
same methods described for in Experiment 1. Twelve of 16 mice had no place
preference after saline conditioning and continued onto the second phase of
the study (N=6 mice/group).</p><!><p>GSK3β is activated in response to cocaine memory reactivation
as evidenced by its dephosphorylation (Shi
et al., 2014). Experiment 3 tested whether the regulation of
GSK3β activity is dependent on GluN2A-containing NMDA receptors. 32
mice underwent cocaine conditioned place preference and testing on Day 9. 23
mice expressed a cocaine place preference and were included for study on Day
10. On Day 10, two groups of mice (N=5–7 mice/group) were injected
with the GluN2A-preferring antagonist NVP-AAM077 (5 mg/kg) or vehicle 30
minutes prior to re-exposure to the compartment previously paired with
cocaine. Two other groups (N=5–6 mice/group) were kept in their home
cages and received NVP-AAM077 or vehicle on the same schedule. Immediately
following the 10 minute reactivation session, mice were exposed to
CO2 for 15 seconds and decapitated. Brains were removed, and
the amygdala, nucleus accumbens, and hippocampus were dissected and prepared
for measurements of phosphoproteins by immunoblotting as described
above.</p><!><p>32 mice underwent 8-day cocaine conditioned place conditioning and
testing on Day 9; 26 mice showed cocaine place preference and continued onto
Day 10. On Day 10, two groups of mice (N=6–7 mice/group) received the
selective GluN2B antagonist ifenprodil (5 mg/kg) or vehicle 30 minutes prior
to re-exposure to the compartment previously paired with cocaine. Two other
groups of mice (N=6–7 mice/group) remained in their home cages and
received ifenprodil or vehicle according to the same schedule. Immediately
following the 10 minute reactivation session, mice were exposed to
CO2 for 15 seconds, decapitated and brains dissected on ice.
The amygdala, nucleus accumbens, and hippocampus from individual mice were
prepared for immunoblotting.</p><!><p>This experiment investigated the role of PPI in the activation of
GSK3β in response to recall of cocaine-related memories using a
pharmacological inhibitor of PP1, okadaic acid. Cannula were permanently
implanted into the lateral ventricle of 38 mice as described above, and mice
had 5–7 days recovery before place conditioning began. 26 mice
expressed a cocaine place preference on Day 9 and were included in Day 10 of
the study, assigned to four groups (N=6–7 mice/group). Experimental
methods are exactly as described in Experiment 3 except that saline or
okadaic acid (150 ng/3 Î¼l, i.c.v.) was administered 30 min prior to
the 10-min reactivation session or in the home cage. Immediately following
the reactivation session, brains were removed and the nucleus accumbens and
hippocampus were dissected and prepared for measurements of phosphoproteins
by immunoblotting.</p><!><p>Cannulas were surgically implanted into the lateral ventricles of 43
mice. After 5–7 days recovery, mice underwent cocaine place
conditioning and testing for place preference on Day 9. Injection cannula in
two mice became dislodged during place conditioning, and one mouse was
unhealthy, so these mice were removed from the study. 29 mice expressed
cocaine place preference on Day 9 and continued on in the study. On Day 10,
two groups of mice (N=7 mice/group) received saline or okadaic acid (150
ng/3 Î¼l, i.c.v.) and 30 min later, were placed into the compartment
previously paired with cocaine for 10 min. Two other groups (N=7–8
mice/group) were kept in their home cages and received saline or okadaic
acid (150 ng/3 Î¼l, i.c.v.) on the same schedule. On Day 11, mice were
re-tested for place preference in order to determine if inhibition of PP1
prior to memory reactivation could attenuate the previously established
cocaine place preference.</p><!><p>Behavioral data (primary endpoint preference scores) were analyzed using
two-way repeated measures analysis of variance (RM-ANOVA) with day and treatment
factors (GraphPad Prism 5, La Jolla, CA). The Kolmogorov-Smirnov test (Prism 5)
was used assess the normality of data. All data sets were normally distributed,
thus parametric statistical tests were used. Protein data (p-GSK3β/total
GSK3β) were analyzed by two-way ANOVA with exposure and treatment
factors, followed by Bonferroni test for multiple comparisons. Grubb's
tests were applied to the protein data to identify potential outliers; 3 samples
were identified as outliers and removed from the data set. The graphs are shown
as standard box plots. The ends of the box indicate the upper and lower
quartiles, the line across the box indicates the median (50th
percentile), and the whiskers from the two ends of the box extend to the minimum
and maximum data points. The level of statistical significance was set at
p<0.05 for all tests.</p><!><p>Experiment 1 tested the role of the GluN2A-containing NMDA receptors in
reconsolidation of cocaine-associated memories. As shown in Fig. 2A, administration of the GluN2A-preferring NMDAR
antagonist NVP-AAM077 (NVP) after the memory reactivation session on Day 10 dose
dependently reduced the previously established cocaine place preference in
subsequent tests conducted 24 hours and 7 days later. Repeated measures two-way
ANOVA of preference scores from Days 9, 11, and 18 (Fig 2A) revealed significant main effects of
NVP-AAM077 treatment (F2,30 = 8.04, p < 0.01)
and test day (F2,30 = 6.18, p < 0.01), as
well as a significant treatment × day interaction (F4,30 =
3.14, p < 0.05). Post hoc tests revealed that
administration of NVP-AAM077 (5 mg/kg) immediately following reactivation of
cocaine reward memories significantly attenuated preference for the
cocaine-paired compartment when tested 24 h later (p <
0.01 vs. vehicle, Day 11) or one week later (p < 0.05
vs. vehicle, Day 18). No significant differences were found between the mice
injected with the vehicle and 1 mg/kg of NVP-AAM077 (Fig. 2A). In the control experiment with no
reactivation session on Day 10, NVP-AAM077 (5 mg/kg) administered in the home
cage had no effect on the previously established cocaine place preference (Fig. 2B). Two-way RM-ANOVA of preference
scores showed no significant effects of NVP-AAM077 treatment (F1,18 =
1.20, p > 0.05), test day (F2,18 = 0.43,
p > 0.05), or treatment×day interaction
(F2,18 = 0.90, p > 0.05).</p><p>Since mice administered 5 mg/kg NVP-AAM077 on Day 10 after memory
reactivation showed negative place preference scores when retreated on Days 11
and 18 (Fig 2A), a second control
experiment was performed to test whether NVP-AAM077 alone could produce a place
aversion under the conditions used in this study. To this end, two additional
groups of mice underwent 8 days of conditioning with saline administered in both
compartments, and both groups showed no place preference on Day 9 (Fig. 2C). Mice were re-exposed to the chamber
for 10 min on Day 10 and received NVP-AAM077 (5 mg/kg) immediately thereafter.
Place preference was re-tested on Days 11 and 18. Two-way ANOVA of preference
scores showed no significant effects of NVP-AAM077 (F1,20 = 0.02,
p > 0.05), test day (F2,20 = 0.54,
p > 0.05), or treatment×day interaction
(F2,20 = 0.09, p > 0.05). Thus,
NVP-AAM077 alone did not cause a significant place aversion but rather
eliminated a place preference produced by prior cocaine conditioning.</p><!><p>Experiment 2 tested the role of GluN2B-containing NMDAR in the
reconsolidation of cocaine-associated memories using ifenprodil. Results
demonstrate that administration of ifenprodil after memory reactivation
dose-dependently attenuated the previous established cocaine place preference in
subsequent tests conducted 24 hours and 7 days later (Fig. 3A). Two-way ANOVA showed significant main
effects of ifenprodil treatment (F3,54 = 7.83, p
< 0.001) and test day (F2,54= 17.58, p
< 0.001), as well as a treatment×day interaction (F6,54
= 8.13, p < 0.001). Post hoc tests revealed that
administration of ifenprodil (5 and 10 mg/kg) immediately after the 10-min
reactivation session significantly attenuated preference for the cocaine-paired
compartment when tested 24 hours later and one week later (p
< 0.01 vs. vehicle for 5 mg/kg; p < 0.001 vs.
vehicle for 10 mg/kg, Days 11 and 18) as shown in Fig. 3A. In contrast, administration of ifenprodil (10 mg/kg, i.p.)
in the home-cage environment had no effect on cocaine place preference (Fig. 3B); two-way ANOVA showed no significant
effects of ifenprodil treatment (F1,21 = 0.18, p
> 0.05), test day (F2,21 = 0.07, p >
0.05), or treatment×day interaction (F2,21 = 0.06,
p > 0.05) in the absence of memory reactivation
(Fig. 3B). When ifenprodil was
administered to mice that had undergone conditioning with saline in both
compartments, no significant effects of ifenprodil treatment (F2,20 =
0.004, p > 0.05), test day (F2,20 = 0.55,
p > 0.05), or treatment×day
interaction (F2,20 = 0.05, p > 0.05) were
found (Fig. 3C). These results show that
ifenprodil was effective in abrogating a cocaine place preference only when
administered after memory reactivation, suggesting that reconsolidation of
cocaine-associated reward memories involves GluN2B-containing NMDA
receptors.</p><!><p>Our prior study demonstrated that GSK3β was activated after
re-exposure to the environment previously paired with cocaine, as evidenced by a
significant reduction in the level of GSK3β phosphorylation (Shi et al., 2014). Experiments 3 and 4
tested whether GSK3β activation was dependent on NMDA receptor activation
and which NMDA receptor subtype might be involved. In experiment 3, mice
underwent cocaine place conditioning and preference testing (Day 9). On Day 10,
mice were pretreated with saline or NVP-AAM077 (5 mg/kg) 30 min prior to the
10-min memory reactivation session (i.e., exposure to the environment previously
paired with cocaine) or in home cages (i.e., no exposure group). Brains were
obtained immediately after 10 min reactivation to investigate the signaling
pathway involved in cocaine memory reactivation. Representative immunoblots of
amygdala (Fig. 4A), nucleus accumbens
(Fig. 4B) and hippocampus (Fig. 4C) from mice with or without exposure
to the environment previously paired with cocaine are presented. Two-way ANOVA
of levels of phospho-GSK3β:GSK3β in amygdala (Fig. 4A) revealed significant main effects of
NVP-AAM077 (F1,19 = 4.63, p < 0.05) and
environment (F1,19 = 8.16, p < 0.05).
Similarly, two-way ANOVA of levels of phospho-GSK3β:GSK3β also
revealed a significant main effect of NVP treatment in the nucleus accumbens
(F1,16 = 15.74, p < 0.01) and hippocampus
(F1,19 = 6.03, p < 0.05), as well as a
treatment×environment interaction in the nucleus accumbens
(F1,16 = 4.61, p < 0.05). Post hoc tests
revealed that levels of phosphorylated GSK3β-Ser9 were lower following
the reactivation of cocaine cue memories compared with no exposure in the
amygdala, nucleus accumbens and hippocampus (all p <
0.05). Mice pretreated with NVP-AAM077 prior to re-exposure to cocaine-paired
environment showed significantly higher levels of p-GSK3β in the amygdala
(p < 0.05), nucleus accumbens (p
< 0.01), and hippocampus (p < 0.05) compared with
saline-injected exposed mice. These data indicate that reactivation of cocaine
memory activates GSK3β (i.e., reduces phospho-GSK3β), which is
dependent on the activation of GluN2A receptors. No differences in levels of
total GSK3β:tubulin were found in any brain region (data not shown).</p><!><p>Mice underwent the same procedures as described in Experiment 3, except
that on Day 10, mice were pretreated with saline or ifenprodil (5 mg/kg) 30 min
prior to the 10-min memory reactivation session or remained in home cages (i.e.,
no exposure group). Brains were obtained immediately after the 10 min
reactivation to investigate the regulation of GSK3β activity during
cocaine memory reactivation. Representative immunoblots of the amygdala (Fig. 5A), nucleus accumbens (Fig. 5B) and hippocampus (Fig. 5C) from mice with or without exposure to the
environment previously paired with cocaine are presented. Two-way ANOVA of
levels of phospho-GSK3β in the amygdala (Fig. 5A) revealed significant main effects of environment
(F1,20 = 9.33, p < 0.01) and ifenprodil
treatment×environment interaction (F1,17 = 6.0,
p < 0.05). Similarly, two-way ANOVA of levels of
phospho-GSK3β also revealed significant main effects of ifenprodil
treatment (F1,20 = 6.40, p < 0.05), as well
as ifenprodil treatment×environment interaction (F1,17 = 4.55,
p < 0.05) in the nucleus accumbens(Fig. 5B). A significant main effect of environment was
found in the hippocampus (F1,20 = 4.78, p <
0.05), as well as ifenprodil treatment×environment interaction
(F1,17 = 10.21, p < 0.01) (Fig. 5C). Post hoc tests revealed that levels of
phosphorylated GSK3β-Ser9 were significantly lower following the
reactivation of cocaine cue memories compared with no exposure in the amygdala,
nucleus accumbens, and hippocampus (all p < 0.01). Mice
pretreated with ifenprodil prior to re-exposure to cocaine-paired environment
showed significantly higher levels of p-GSK3β in the amygdala
(p < 0.05), nucleus accumbens (p
< 0.01), and hippocampus (p < 0.01) compared with
saline-injected exposed mice. These data indicate that reactivation of cocaine
memory activates GSK3β (i.e., reduces p-GSK3β), which is dependent
on activity of GluN2B receptors.</p><!><p>Experiment 5 investigated whether activation of GSK3β produced by
recall of cocaine reward memories is dependent on protein phosphatase1 (PP1)
function. Following cocaine place conditioning and preference testing (Day 9),
two groups of mice were pretreated with saline or okadaic acid (OA; 150 ng/3ul,
i.c.v) 30 min prior to the 10-min reactivation session on Day 10 (i.e.,
re-exposure groups). Two other groups remained in their home cages and received
the same pretreatment without memory reactivation (i.e., no exposure groups).
Brains were obtained immediately thereafter. Representative immunoblots of the
nucleus accumbens (Fig. 6A) and hippocampus
(Fig. 6B) from mice with or without
exposure to the environment previously paired with cocaine are presented.
Two-way ANOVA of levels of phospho-GSK3β in the nucleus accumbens (Fig. 6A) revealed significant main effects of
okadaic acid treatment (F1,21 = 22.04, p <
0.001) and exposure condition (F1,21 = 7.71, p
< 0.05). Similarly, two-way ANOVA of levels of phospho-GSK3β in
the hippocampus also revealed significant main effects of okadaic treatment
(F1,21 = 23.77, p < 0.001) and exposure
condition (F1,21 = 10.53, p < 0.01). Post hoc
tests revealed that okadaic acid significantly increased the levels of
phospho-GSK3β in both the nucleus accumbens and hippocampus
(p < 0.05 vs saline) under the 'No
Exposure' condition. Reactivation of cocaine cue memories resulted in
lower levels of phospho-GSK3β in the nucleus accumbens
(p < 0.05; saline no exposure vs saline exposure)
and hippocampus (p < 0.05). Compared with saline
pretreatment, okadaic acid administration prior to exposure to cocaine-paired
environment resulted in significantly higher levels of phospho-GSK3β in
the nucleus accumbens (p < 0.01 saline exposure vs OA
exposure) and hippocampus (p < 0.001 saline exposure vs
OA exposure). These data suggest that PP1 regulates the phosphorylation of
GSK3β, including during recall of cocaine-associated memories.</p><!><p>Since the activation of GSK3β induced by re-exposure to an
environment previously associated with cocaine was blocked by PP1 inhibition,
the ability of okadaic acid to interfere with the reconsolidation of cocaine
reward memories was investigated. Following an 8-day cocaine conditioning
procedure, two groups of mice showed similar preferences for the cocaine-paired
compartment of the conditioning chamber on Day 9 (Fig. 7). On Day 10, mice were pretreated with okadaic acid (150
ng/3ul, i.c.v) or saline 30 minutes before confinement to the prior
cocaine-paired compartment. After the 10-minute re-exposure, mice were returned
to the home cage and preference was tested again 24 hours later. Two-way
RM-ANOVA of preference scores on Days 9 and 11 revealed significant main effects
of okadaic acid treatment (F1,22= 6.91, p <
0.05) and test day (F1,22 = 6.0739, p <
0.05), and a treatment×day interaction (F1,22 = 4.867,
p < 0.05). Post hoc tests revealed that
administration of okadaic acid prior to cocaine memory reactivation abolished
the previously established preference for the cocaine-paired compartment when
tested 24 h later (p < 0.01 vs. saline, Day 11. Fig. 7A). Two additional groups of mice
underwent similar cocaine place conditioning and testing on Day 9. On Day 10,
these mice were injected with vehicle or okadaic acid (150 ng/3ul, i.c.v) in
their home cage without re-exposure to the cocaine-paired environment. When
preference was re-tested 24 hours later, both groups of mice maintained their
cocaine place preference at levels similar to Day 9 and were not significantly
different from each other. Two-way RM-ANOVA of preference scores showed no
significant effects of okadaic acide treatement
(F1,13 = 0.36,
p > 0.05), test day
(F1,13 = 0.53,
p > 0.05), or treatment×day
interaction (F1,13 = 0.003,
p > 0.05) (Fig. 7B).</p><!><p>Our prior work demonstrated the requirement of GSK3β signaling in the
reconsolidation of cocaine reward memories. GSK3β is activated by
reactivation of cocaine-associated contextual memories, and inhibition of
GSK3β after memory reactivation prevents reconsolidation and erases a
previously established cocaine place preference (Shi
et al., 2014). The present study sought to determine the upstream factors
that control GSK3β activity and are critical for the maintenance of cocaine
reward memory. Results presented herein demonstrate the importance of GluN2A and
GluN2B NMDA receptor subtype activation during recall of cocaine reward memory to
the regulation of GSK3β activity and the maintenance of these memories.
Further, the data identify brain regions of potential significance for the
reconsolidation of cocaine reward memory.</p><p>Previous studies have demonstrated the importance of NMDA receptors in the
reconsolidation process (Alaghband & Marshall
2013; Itzhak 2008; Liddie & Itzhak 2016). NMDA receptors are heteromeric
complexes composed of two GluN1 and two GluN2 subunits. There are four different
GluN2 subunits, with GluN2A and 2B implicated in learning and memory (Ben Mamou et al. 2006; Milton et al. 2013; Shipton & Paulsen 2014). The present study
investigated the roles of GluN2A- and GluN2B-containing NMDA receptors in
reconsolidation of cocaine memories. Results demonstrate that blockade of
GluN2A-containing NMDA receptors with the antagonist, NVP-AAM077, disrupted cocaine
memory reconsolidation as evidenced by loss of a previously established cocaine
place preference when tested 24 hours and 7 days later. Further, GluN2A receptors
were found to regulate GSK3β activity during memory reactivation. Blockade of
GluN2A receptors prior to cocaine memory reactivation prevented the
dephosphorylation of GSK3β in the amygdala, nucleus accumbens and
hippocampus, indicating that GluN2A-containing receptors are involved in the
activation of GSK3β following reactivation of cocaine reward memory. Upon
investigating the role of GluN2B-containing NMDA receptors in reconsolidation of
cocaine reward memories, the selective GluN2B antagonist ifenprodil (Brittain et al. 2012) was found to
attenuate a previously established cocaine place preference when administered
immediately after memory reactivation. Ifenprodil also prevented the
dephosphorylation of GSK3β in the amygdala, nucleus accumbens and
hippocampus, indicating that GluN2B receptors are involved in the activation of
GSK3β following cocaine reward memory reactivation.</p><p>The ability of NVP-AAM077 or ifenprodil to disrupt cocaine mnemonic traces
only occurred when these agents were administered following memory reactivation,
providing further support that they are specifically interfering with
reconsolidation of cocaine-associated memories rather than impairing memory recall.
Further support for interference with reconsolidation comes from the finding that
there was no spontaneous recovery of place preference 7 days later. Our findings
suggest that both GluN2A- and GluN2B-containing NMDA receptors are required for the
reconsolidation of cocaine contextual memories. However, a potential caveat to this
conclusion involves consideration of the selectivity of the GluN2A inhibitor. The
relative selectivity of NVP-AAM077 for the GluN2A subunit over the GluN2B subunit is
not as high as originally suggested (Auberson
et al. 2002; Frizelle et al. 2006), and so NVP-AAM077 may be a
preferential rather than selective GluN2A antagonist (Berberich et al. 2005). Thus, it cannot be ruled out
that some of the effects of NVP-AAM077 on the reconsolidation of cocaine-associated
memories reported here are mediated by GluN2B-containing receptors. The present
findings with ifenprodil support a prior study demonstrating that ifenprodil or
traxoprodil administered after reactivation of cocaine contextual memories disrupts
an established place preferences (Liddie &
Itzhak 2016), and so together demonstrate the importance of
GluN2B-containing NMDA receptors in the reconsolidation of cocaine reward
memories.</p><p>The regulation of GluN2A- and Glu2B-NMDA receptors during the
reconsolidation of cocaine memories associated with cocaine self-administration has
been investigated to a limited extent. Phosphorylation of GluN2A, but not GluN2B, in
the dorsal hippocampus is higher during recall of cocaine contextual memories
associated with the drug self-administration chamber (Wells et al. 2016). In addition,
antagonism of GluN2A-containing NMDA receptors by NVP-AAM or PEAQX following
cocaine-memory reactivation impairs subsequent drug context-induced cocaine-seeking
(Wells et al., 2016). This report did not
include a GluN2B antagonist, but it supports a role for GluN2A-containing receptors
in reconsolidation of cocaine contextual memories as associated with cocaine
self-administration.</p><p>In addition to reward memories, the involvement of subtypes of NMDA
receptors in reconsolidation of aversive memory has been reported. GluN2A-containing
(Milton et al, 2013), but not
GluN2B-containing (Ben Mamou et al, 2006) NMDA
receptors are required for fear memory reconsolidation, whereas GluN2B receptors are
involved in destabilization of reactivated memories (Ben Mamou et al. 2006; Milton et al. 2013). However, inconsistencies in the
literature exist. Nakayama and colleagues showed that GluN2B NMDA receptor signaling
may be involved in both the restabilization of reactivated memories and their
destabilization (Nakayama et al.
2016). The disparate results across studies may be due to differences in
experimental design including site and timing of drug injection (Ben Mamou et al. 2006; Nakayama et al. 2016).</p><p>The present study also investigated PP1 which is known to be an activator of
GSK3β via dephosphorylation of Ser9-GSK3β (Peineau et al. 2008; Szatmari et al. 2005). NMDA receptor
stimulation in both cultured primary (cortical and hippocampal) neurons and adult
mouse brain (striatum and hippocampus) rapidly activates GSK3β by
dephosphorylation of Ser9-GSK3β (Szatmari et
al, 2005). Further, dephosphorylation of GSK3β triggered by NMDA
receptor stimulation is inhibited by 1Î¼M okadaic acid, a concentration that
inhibits both PP1 and PP2A (Cohen et
al. 1989). At PP2A-selective concentrations, okadaic acid
fails to affect NMDA regulation of Ser9-GSK3β (Szatmari et al, 2005), suggesting that PP1 is responsible
for the dephosphorylation of GSK3β. Our previous study showed that levels of
phospho-GSK3β were downregulated following reactivation of cocaine reward
memory, and inhibition of GSK3 disrupted cocaine memory reconsolidation (Shi et al, 2014). In the present study, okadaic
acid administered prior to cocaine memory reactivation blocked the dephosphorylation
of GSK3β, indicating that activation of GSK3β following recall of
cocaine memory is dependent on PP1. Okadaic acid increased the levels of
Ser9-GSK3β phosphorylation in animals without memory reactivation, so PP1 may
be involved in regulating phospho-GSK3β levels in this basal condition.</p><!><p>When established memories are reactivated by recall, they become labile and
susceptible to disruption. This provides an opportunity to interfere with the
maintenance of memory traces that contribute to pathological states, in this case
drug-seeking behaviors. The data presented here suggest that following reactivation
of cocaine reward memories, GluN2A- and GluN2B-containing NMDA receptors are
stimulated, PP1 is activated and the activity of GSK3β is induced
accordingly. Further, the function of this pathway is necessary for the
reconsolidation of a cocaine reward mnemonic trace; inhibition of this signaling
pathway at any step is sufficient to impair the maintenance of such memories.
Post-reactivation inhibition of GluN2A- and GluN2B-containing NMDA receptor subtypes
impairs reconsolidation and erases an established cocaine place preference, thus
making them potential therapeutic targets in treatment of cocaine dependence and the
prevention of relapse.</p>