Measurement of corticosterone levels

Plasma corticosterone levels were determined before and after FST. One hundred
microliters whole blood was collected from mouse superficial temporal vein. The vein
was punctured using a 26G needle and blood was collected in heparin coated capillary
tubes. Upon completion, the wound was gently patted with cotton to stop bleeding. Mice
were then returned to their home cage. Samples were centrifuged (10min; 2000xg) and
plasma corticosterone levels were quantified using a corticosterone enzymeimmunoassay
kit (immunodiagnostic systems, Scottsdale AZ). The pre-FST blood was collected at the
end of extinction test (Day 15) while the post-FST blood was collected following CPP
test on Day 16. Pre- and post-FST blood samples were collected from opposite sides of
the face. Since plasma corticosterone levels follow a circadian pattern of secretion
(Malisch et al., 2008), blood samples were collected at the same time each day.
Statistical Analysis
Data for CPP testing and immobility during forced swim were analyzed using
two-way repeated measures ANOVA (group x time) followed by Student-Newman-Keuls
post hoc test. Two-way ANOVA (drug treatment x session) was used to compare pre- and
post-FST plasma corticosterone levels. One-way repeated measures ANOVA followed by
Holm-Sidak post hoc test was used to compare differences in CPP magnitude for no-stress
controls. Data analysis was done using Sigma Stat version 3.1 (Systat Software Inc.).
Results
Stress-induced reinstatement of Fix-C and Esc-C CPP
a) Effect of MK-801
A test of place preference following extinction training (day 15) showed that mice
displayed a reduction in preference for the previously cocaine-paired compartment (Fig.
4.1A, B). We then investigated the effect of an acute injection of MK-801 on forced
swim-induced reinstatement of Fix-C and Esc-C CPP. While vehicle-treated mice
subjected to FST showed significant recovery of CPP, administration of MK-801 prior to
FST-1 significantly attenuated stress-induced reinstatement in Fix-C but not Esc-C mice
(day 16). Additionally, exposure to a subsequent FST trial where mice were not under the
influence of MK-801 failed to reinstate CPP to vehicle control levels (day 18) in Fix-C
but not Esc-C. Two-way repeated measures ANOVA showed a significant time effect
[for Fix-C: F(5,65)=8.768; p<0.001); for Esc-C: F(5,65)=9.959; p<0.001] but no significant
group effect [for Fix-C: F(1,65)=4.187; p=0.062); for Esc-C: F(1,65)=1.169; p=0.299] and no
significant group x time interaction (p>0.05). Post hoc analysis showed that for Fix-C,
the magnitude of place preference in MK-801-treated mice was significantly attenuated
compared to vehicle-treated mice on days 16 (p=0.024) and 18 (p=0.016). Importantly,
mice not subjected to the FST did not show spontaneous recovery of place preference on
days 16 and 18 (Fig. 4.5). One-way repeated measures ANOVA showed a significant
time effect (F(5,40)=55.824; p<0.001). Post hoc analysis showed that the magnitude of
place preference after conditioning (day 8) was significantly higher compared to all other
test days (in all cases p<0.001). Together, these results suggest differential effects of MK-
801 on stress-induced reinstatement of Fix-C and Esc-C CPP; MK-801 attenuated
reinstatement of Fix-C CPP but had no effect on reinstatement of Esc-C CPP.
b) Effect of the NR2B antagonist ifenprodil
Systemic administration of the NR2B-containing NMDAR antagonist ifenprodil
had no effect on stress-induced reinstatement of Fix-C and Esc-C CPP (Fig. 4.1C, D).
Two-way repeated measures ANOVA showed a significant time effect [for Fix-C:
F(5,80)=15.546; p<0.001; for Esc-C: F(5,85)=13.668; p<0.001] but no significant group
effects (p>0.05) and no significant group x time interaction (p>0.05). Results suggest that
there is no specific requirement for the NR2B subunit of NMDAR in stress-induced
reinstatement of Fix-C and Esc-C CPP.
c) Effect of the nNOS inhibitor 7-NI
We next investigated whether inhibition of signaling molecules downstream of
the NMDAR would attenuate stress-induced reinstatement of Fix-C and Esc-C CPP. The
nNOS inhibitor 7-NI disrupted reinstatement of Fix-C but not Esc-C CPP (Fig. 4.2). For
Fix-C, two-way repeated measures ANOVA showed a significant overall group effect
(F(1,110)=11.597; p=0.003), a significant time effect (F(5,110)=10.851; p<0.001) and a
significant group x time interaction (F(5,110)=3.928; p=0.003). Post hoc analysis showed
that 7-NI treated animals displayed significantly lower place preference compared to
vehicle-treated animals when subjected to FST on days 16 (p<0.001) and 18 (p=0.005).
On the other hand, inhibition of nNOS had no effect on stress-induced reinstatement of
Esc-C CPP. Two-way repeated measures ANOVA showed there was a significant time
effect (F(5,85)=18.588; p<0.001) but no significant group effect (p>0.05) and no
significant group x time interaction (p>0.05). Results suggest that while Fix-C CPP
engages NO-signaling, stress-induced reinstatement of Esc-C CPP is insensitive to
manipulation of NO-signaling.
Additionally, 7-NI administration prior to FST did not affect mobility in
subsequent FST trials (Fig. 4.3). A two-way repeated measures ANOVA shows a
significant time effect [F(4,44)=8.789; p<0.001] but no significant group effect
[F(1,44)=0.00147; p=0.97] and no significant group x time interaction (p>0.05).
d) Effect of the CRH-R1 antagonist antalarmin
To determine the contribution of CRH receptor subtype 1 to stress-induced
reinstatement of Fix-C and Esc-C CPP, we administered antalarmin 30 min prior to
exposure to FST-1. Antalarmin attenuated stress-induced reinstatement of Fix-C but not
Esc-C CPP (Fig. 4.4). With respect to Fix-C, two-way repeated measures ANOVA
showed a significant time effect [F(5,50)=2.635; p=0.034] but no significant group effect
[F(1,50)=3.416; p=0.094] and no significant group x time effect [F(5,50)=1.285; p=0.285].
However, post hoc analysis showed that antalarmin-treated mice showed significantly
reduced magnitude of place preference compared to vehicle-treated animals on day 16.
There was a non-significant trend toward a reduction in CPP magnitude for antalarmin74
treated animals following the second forced swim test on day 18 (Fig. 4.4A). With
respect to Esc-C CPP, two-way repeated measures ANOVA showed a significant time
effect [F(5,65)=29.951; p<0.001] but no significant group effect (p>0.05) and no
significant group x time interaction (p>0.05). Antalarmin-treated mice showed similar
levels of place preference compared to vehicle-treated mice following exposure to two
FST trials (days 16 and 18; Fig. 4.4B).
Plasma corticosterone levels
Plasma corticosterone levels measured following FST were approximately 7-10
fold higher compared to corticosterone levels prior to exposure to FST (Fig. 4.6). Twoway
ANOVA (treatment x session) analysis of levels of corticosterone before and after
FST showed there was a significant effect of test session (F(1,26)=273.242; p<0.001).
However, there was no significant treatment effect as pre-FST administration of
antalarmin, MK-801 or 7-NI showed similar levels of plasma corticosterone as vehicletreated
animals (F(3,26)=0.306; p=0.821). Additionally, there was no significant treatment
x test session effect (p>0.05).


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