CPP Test, Extinction and reinstatement, Data Analysis

The expression of place preference was tested 3 days after the final training
session in order to eliminate possible contributions from residual cocaine administration
on the last training session and also to allow the consolidation of long-term memory of
drug reward. Similar to habituation, CPP testing was carried out between 12:00 and 14:00
hours.
Extinction and reinstatement
Conditioned mice were allowed free exploration for 20 minutes in the training
context 24 hours after the CPP test. Since we aimed to facilitate the consolidation of
extinction, immediately following each extinction session mice received single IP
injections of either a) vehicle (n=5; n=8)/saline (n=5), b) rolipram (0.25mg/kg, n=6), c)
BAY-73-6691 (3mg/kg, n=10), or d) Papaverine (20mg/kg, n=6) and were returned to
their home cages. Rates of extinction, herein defined as a significant reduction in the
magnitude of CPP compared to CPP test, were recorded daily. Mice received a two-day
break from extinction training after 4 days. Training resumed the following day and
continued for the next 3 days (Table 3.1).
Twenty-four hours after the last extinction session, mice received a priming dose
of cocaine (12mg/kg; the average daily dose during conditioning) in the absence of the
PDE inhibitor. Time spent in each compartment as well as locomotor activity was
recorded for 20 minutes.
Data Analysis
Changes in levels of cyclic nucleotides in response to different doses of a PDE
inhibitor were analyzed by one-way ANOVA supplemented by Holm-Sidak method for
post-hoc. Results of CPP are presented as mean ± SEM of the difference in time spent in
cocaine- and saline-paired compartments. The overall effect on acquisition, extinction
and reinstatement of cocaine-induced place preference was analyzed by two-way
ANOVA (group X time) general linear model supplemented with post-hoc multiple pairwise
comparisons using the Tukey Test. Further, unpaired Student t-test or Mann-
Whitney rank sum test where appropriate were used to compare differences in the
magnitude of CPP upon cocaine priming versus the final day of extinction training. For
all measures, significance was considered from a value of p<0.05.
Results
Effects of PDE inhibitors on cAMP and cGMP levels
The PDE4 inhibitor rolipram dose dependently increased levels of cAMP in both
the hippocampus and amygdala (Figure 3.1A) but had no effect on cGMP levels (data not
shown). Rolipram treatment resulted in an overall significant increase in cAMP in both
the hippocampus (F[3,8]=18.463; p<0.001) and amygdala (F[3,8]=17.824; P<0.001). At
doses of 0.25 and 1mg/kg rolipram administration resulted in about 2-fold increase in
cAMP in the amygdala (t=6.102; p<0.001 and t=6.166; p<0.001, respectively) and
hippocampus (t=6.072; p<0.001 and t=4.858; p<0.001, respectively) compared to vehicle
controls; a dose of 0.05mg/kg rolipram also significantly increased levels of cAMP in the
amygdala (t=2.606; p=0.031). A dose of 0.25mg/kg rolipram was used for the subsequent
behavioral experiment. The PDE9 inhibitor BAY-73-6691 dose dependently increased
levels of cGMP in both the hippocampus (F[3,8]=27.052; p<0.001) and amygdala
(F[3,8]=7.941; p<0.001) (Fig 3.1C) but had no effect on levels of cAMP (data not shown).
A dose of 3mg/kg of BAY-73-6691 was used in the extinction experiments because this
dose resulted in the highest increase in levels of cGMP (t=4.208; p=0.003 and t=8.549;
p<0.001) for amygdala and hippocampus, respectively. Significant increases in cGMP
were also observed in the hippocampus for dosages of 0.03mg/kg (t=3.157; p=0.013) and
0.3mg/kg (t=5.969; p<0.001) and amygdala at 0.3mg/kg (t=3.272; p=0.011). An overall
significant effect of papaverine on levels of cAMP was observed in the hippocampus
(F[3,8]=6.531; p=0.015) but not in the amygdala (F[3,9]=1.225; p=0.356). Significant
increases in cGMP in both the hippocampus (F[3,10]=7.613; p=0.006) and amygdala
(F[3,10]=4.929; p=0.024) were observed. Holm-Sidak post-hoc analysis showed that the
PDE10A inhibitor papaverine at10, 20 and 40mg/kg significantly increased levels of
cAMP in the hippocampus (t=3.997; p=0.004, t=3.640; p=0.007, and t=2.674; p=0.028,
respectively) but not in the amygdala (Fig. 3.1B). However, post-hoc comparisons
showed that papaverine increased cGMP in both the hippocampus at 10, 20 and 40mg/kg
(t=2.984; p=0.014, t=4.286; p=0.002 and t=4.077; p=0.002, respectively) and the
amygdala at 40mg/kg (t=3.715; p=0.004) (Fig. 3.1D). A dose of 20mg/kg was chosen for
further experiments because a) unpaired Student t-test showed a significant difference
between controls and mice that received 20mg/kg papaverine (t[5]=-9.542; p<0.001) and
b) the high dose of 40mg/kg resulted in ataxia in mice. Differences in control levels of
cAMP in Fig. 3.1A compared to Fig. 3.1B may have arisen because two different
vehicles were used, 2% DMSO for rolipram experiment versus saline for papaverine
experiment. Similarly, in Fig. 3.1C, the vehicle for BAY-73-6691 was Solutol HS 15
while that for papaverine in Fig. 3.1D was saline.
Extinction of cocaine-induced place preference
Mice that received saline or vehicle during extinction training did not show
“extinction” (Fig. 3.2). These results are consistent with a previous study showing that
conditioning by escalating doses of cocaine resulted in extinction-resistant CPP (Itzhak &
Anderson, 2011). Administration of rolipram immediately after each extinction session
for a total of 8 days had no effect on the magnitude of place preference compared to the
vehicle group (Figure 3.2A). In a separate experiment, prolonged extinction training (12
days) followed by rolipram administration had also no effect (data not shown). Since
there was no evidence of extinction in both groups, a test for reinstatement was not
performed.
Administration of BAY-73-6691 immediately after each extinction session
resulted in significant reduction in the magnitude of CPP compared to controls
(F[1,176]=12.168; p<0.001, two-way ANOVA) (Fig. 3.2B). Post-hoc comparison by Tukey
test showed significant differences between the two groups on days 10 (q=3.307;
p=0.019), 11 (q=3.402; p=0.016) and 12 (q=3.932; p=0.005). On day 11, the difference
between times spent in drug- and saline-paired compartments was 95+162 seconds (Fig.
3.2B), suggesting extinction of CPP since this was significantly different from CPP test
(T=133; p=0.038, Mann-Whitney rank sum test ). BAY-73-6691 also attenuated the
reinstatement of CPP; the difference between the magnitude of CPP on the final day of
extinction (day 11) and the day of cocaine priming (day 12) was not significant (T=90;
56
p=0.273, Mann-Whitney Rank Sum test). However, the vehicle treated group showed a
significant increase in the magnitude of CPP following cocaine priming compared to the
final day of extinction (t[14]=-2.314; p=0.038) (Fig. 3.2B). This finding suggests that
BAY-73-6691 may have provided partial resistance against reinstatement of cocaine
CPP. The differences in extinction rate and resistance to reinstatement in mice treated
with BAY-73-6691 versus vehicle control mice were not due to differences in locomotor
behavior. Results of locomotor activity, which was recorded during each session, showed
that during the reinstatement test, ambulatory counts in control and BAY-73-6691 groups
were 1560+143 and 1642+139 counts/20min, respectively.
Finally, administration of papaverine immediately after each extinction session
had no significant effect on extinction (F[1,99]=0.539; p>0.05) (Fig. 3.2C). The magnitude
of CPP in the papaverine group fluctuated over time. Although a reduction in CPP was
observed on days 8-11 (Fig. 3.2C), the differences between the control and the
papaverine group did not reach statistical significance. However, because there was a
trend of reduction in the magnitude of CPP in both groups, challenge cocaine (12mg/kg)
was given on day 12. A significant increase in CPP was observed in the papaverine
treated group (T=98; p<0.003, Mann-Whitney rank sum test) upon cocaine priming.
However, the increase in CPP following a priming injection to the control group was not
statistically significant (Fig. 3.2C).


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