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Experiment 2: Acquisition of Esc-C and Fix-C memory

The effect of the NR2B antagonist ifenprodil on acquisition of place preference
was investigated because of the observed increase in NR2B subunit expression in the
hippocampus. Additionally, because inhibition of nNOS was shown to block Fix-C
memory acquisition (Itzhak et al., 1998) and since nNOS activity is regulated by calcium
entry through NMDAR (Christopherson et al., 1999; Sattler et al., 1999) we investigated
the effect of the nNOS inhibitor 7-NI on acquisition of Fix-C and Esc-C memory. Thirty
minutes prior to each cocaine session, mice received a) vehicle, b) ifenprodil (10mg/kg)
or c) 7-NI (25mg/kg). Mice were tested for place preference (15min) 72hr later (day 8).
Experiment 3: Reconsolidation of Fix-C and Esc-C memory
3a) Effect of the NMDAR antagonist MK-801
Because the NMDAR antagonist MK-801 disrupted reconsolidation of cocaineassociated
memory acquired by a Fix-C schedule (Kelley et al., 2007; Alaghband &
Marshall, 2013), we investigated the effect of MK-801 on reconsolidation of Fix-C and
Esc-C memory. Following conditioning, on day 8, mice (n=6-10/group) received MK-
801 (0.1 or 0.3mg/kg) pre- or post-retrieval of place memory. For pre-retrieval, drug was
given 30 min prior to CPP expression test (15min) and for post-retrieval drug was given
26
following a 6 min CPP expression test. Mice were then tested for place preference
expression after 4 days.
3b) Effect of NR2B subunit antagonists ifenprodil and traxoprodil
Next, we investigated the effects of ifenprodil and traxoprodil on Fix-C and Esc-C
memory reconsolidation. On day 8, mice were re-exposed to the conditioning apparatus
for 6min (memory retrieval by a short CPP test) and immediately thereafter they received
ifenprodil (10mg/kg) or traxoprodil (10mg/kg). To test whether subsequent reductions in
CPP following ifenprodil treatment was due to disruption of memory reconsolidation,
ifenprodil was administered a) in the home cage in the absence of memory retrieval or b)
6h after memory retrieval (outside the reconsolidation window). Mice were then tested
for place preference expression 4 days following the first memory retrieval session.
3c) Effect of the nNOS inhibitor 7-NI and the MEK inhibitor SL327
To investigate the role of NMDAR downstream signaling in memory
reconsolidation, the effects of the nNOS inhibitor 7-NI and the MEK inhibitor SL327
were investigated. Mice were conditioned by Fix-C and Esc-C schedules and after 72h
mice were re-exposed to the conditioning apparartus for 6 min (memory retrieval by a
short CPP test). Vehicle, 7-NI (25mg/kg) or SL327 (30mg/kg) were administered
immediately thereafter. Mice were then tested for place preference expression 4-7 days
following the first memory retrieval session.
Data Analysis
Data for behavioral testing were analyzed using two-way ANOVA [(group x
time) or (MK-801 dose x time; experiment 3)] followed by Student-Newman-Keuls post
hoc test. qPCR data were analyzed using 2-ΔΔCT method as described by Livak and
Schmittgen (2001). Differences in expression levels in qPCR and western blot analyses
were analyzed by one-way ANOVA. Student’s t-test was also used to compare differences
between two groups. Data analysis was done using Sigma Stat version 3.1 (Systat Software
Inc.).
Results
Experiment 1: Contribution of NMDAR subunits in formation of Fix-C and Esc-C
memory
Mice that received cocaine paired to the training context demonstrated significant
place preference while mice that received either saline injections or cocaine unpaired to
the training context did not (Fig. 2.1A; day 6). Two-way ANOVA showed there was a
significant group effect [for Fix-C:F(2,48)=7.422; p=0.002; for Esc-C:F(2,54)=12.258;
p<0.001]; a significant time effect [for Fix-C:F(1,48)=10.201; p=0.002; for Esc-
C:F(1,54)=30.231; p<0.001] and a significant group x time interaction [for Fix-
C:F(2,48)=4.323; p=0.019; for Esc-C:F(2,54)=8.898; p<0.001] . A comparison of the
magnitude of CPP on day 6 between Esc-C-paired and Fix-C-paired found that Esc-C
showed significantly higher CPP than Fix-C (t=2.355; p=0.031) – thus confirming our
previous findings (Itzhak & Anderson, 2012).
With respect to NMDAR subunit mRNA expression, one-way ANOVA comparing
Grin2b expression among the saline control group, and the Fix-C and Esc-C paired and
unpaired groups showed a significant group effect [F(4,15)=6.473; p=0.003; Fig. 2.1B].
Grin2b was significantly up-regulated in the paired Esc-C group compared to all other
groups; no significant difference in Grin2b expression between the unpaired groups (Fix-C
and Esc-C) was observed (Fig. 2.1B). Levels of Grin1 and Grin2a mRNA were unchanged
across all groups (data not shown).
Because the unpaired conditions showed no significant differences in Grin2b
mRNA expression, western blot analyses were conducted using only the paired conditions
(Fix-C and Esc-C). Western blot analyses of NR2A, NR2B and the compulsory NR1
subunits were carried out. NR2B was significantly up-regulated in Esc-C compared to
Fix-C and saline-treated mice. One-way ANOVA revealed an overall significant group
effect [F(2,8)=12.664; p=0.003]. Figure 2.1C shows an approximate 2.5-fold increase in total
NR2B in the hippocampus of Esc-C compared to Fix-C and approximately 5-fold increase
compared to saline-treated mice. Student’s t-test showed Fix-C was also significantly upregulated
approximately 2.3-fold over saline-treated mice (t=2.684; p=0.044). There were
no significant differences in expression levels of NR1 and NR2A across the groups
although there appeared to be a trend toward reduction in NR2A where saline>Fix-C>Esc-
C (Fig. 2.1D, E).

Experiment 2: Acquisition of Esc-C and Fix-C memory
We investigated whether systemic administration of the NR2B-containing
NMDAR antagonist ifenprodil would attenuate the acquisition of Fix-C and Esc-C
memory. Two-way ANOVA analysis of the effect of ifenprodil treatment on cocaineassociated
memory expression showed that for Esc-C, there was a significant group effect
[F(1,30)=6.678; p=0.015], a significant time effect (test day) [F(1,30)=121.882; p<0.001] and
a significant group x time interaction [F(1,30)=7.586; p=0.01]. For Fix-C, there was a
significant time effect [F(1,28)=46.351; p<0.001], however, there was no significant group
effect [F(1,28)=1.894; p=0.18] and no significant group x time interaction [F(1,28)=4.001;
p=0.055]. Post hoc analyses showed that administration of ifenprodil 30 min before each
cocaine administration session significantly reduced the expression of place preference in
both Esc-C (p=0.004) and Fix-C (p=0.024) compared to saline-treated animals (Fig 2.2B,
C; day 8).
We next investigated whether inhibition of nNOS influences acquisition of Esc-C
memory. Pretreatment with the nNOS inhibitor 7-NI blunted the development of place
preference for mice conditioned by Fix-C [F(1,26)=4.908; p=0.036; group effect] but not
Esc-C (p=0.995) (Fig 2.2D, E), suggesting NO-independent formation of Esc-C memory.
There was a significant time effect for both Fix-C [F(1,26)=64.991; p<0.001] and Esc-C
[F(1,26)=150.662; p<0.001], however, there was no significant group x time interaction for
neither Fix-C nor Esc-C groups (p>0.05).

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