Discussion about CPP

The current study employed an escalating dose schedule for cocaine-induced CPP
as opposed to a typical fixed schedule used in other studies. Mice conditioned by
ascending doses of cocaine maintain a higher magnitude of CPP and show greater
resistance to extinction compared to mice conditioned by a fixed daily dose of cocaine
(Itzhak & Anderson, 2012). Furthermore, an increasing dose schedule more closely
resembles human drug use, in which the quantity of drug increases over time. Indeed
results depicted in Fig. 3.2 indicate that cocaine CPP was long-lasting in control groups
that received saline/vehicle during post-extinction training. Along this vein, a PDEi that
successfully extinguishes place preference at its highest degree, and provides resistance
to cocaine reinstatement, may be particularly valuable relative to its effect on extinction
of a weaker conditioned response that results from conditioning by a fixed dose of
The purpose of this study was to determine how different PDEi with differential
specificity for elevating levels of cAMP and cGMP influence extinction learning. Our
studies focused on the effects of the PDEi on the levels of cyclic nucleotides in the
hippocampus and amygdala because these brain regions have a role in spatial and
emotional learning, respectively, associated with the CPP paradigm.
Since PDE4 is highly expressed in the hippocampus, investigation of a specific
PDE4 inhibitor seemed feasible to study consolidation of extinction. Here we show that
although acute injection of rolipram increased levels of cAMP in both the hippocampus
and amygdala, it did not influence the extinction of cocaine-induced place preference.
Treatment with rolipram during or 30-min prior to but not following cocaine
administration attenuated CPP (Thompson et al., 2004) and reduced cocaine-induced
behavioral sensitization (Janes et al., 2009). This suggests that rolipram is not effective in
reducing cocaine effects if given after drug administration. Therefore, it appears that an
increase in cAMP may not facilitate extinction of cocaine CPP. Although the present
study was not focused on determination of compensatory mechanism associated with
repeated rolipram administration, it has been reported that two weeks administration of
rolipram resulted in a 17-fold increase in PDE4D3 and a down-regulation of PDE4A1
(30%) and PDE4A5 (37%) in rat hippocampus (Dlaboga et al., 2006). The latter may also
explain why rolipram did not induce the extinction of place preference.
We next investigated whether selective increases in brain cGMP facilitate
extinction learning. BAY-73-6691 is a selective inhibitor of PDE9 and may be a potential
therapeutic for Alzheimer’s disease (Bender & Beavo, 2006). PDE9 is one of the most
recently discovered PDE families with the highest affinity for cGMP. In rat and mouse
brain, high densities of PDE9 were found in regions containing soluble guanlyl cyclase
(sGC) and neuronal nitric oxide synthase (nNOS) including the hippocampus, amygdala,
olfactory bulb, allocortex and basal ganglia (Andreeva et al., 2001; van Staveren et al.,
2002). Additionally, BAY-73-6691 has been shown to improve learning and memory in
rodents (van der Staay et al., 2008).
In the present study, acute injection of BAY-73-6691 increased levels of cGMP in
the hippocampus and amygdala, induced extinction of cocaine-induced place preference
and attenuated place preference reinstatement upon cocaine priming. A plausible
explanation for BAY-73-6691 accelerating extinction learning is the involvement of
cGMP in the NO/sGC/PKG/CREB signaling pathway. It has been reported that elevated
cGMP levels in the hippocampus and amygdala, brain regions known to be important for
learning and memory, enhances synaptic plasticity through NO which acts both pre- and
post-synaptically to promote LTP (Arancio et al., 2001; Kleppisch & Feil, 2009; Lu et
al., 1999; Ota et al., 2010; Son et al., 1998).
The PDE5 inhibitor sildenafil (Viagra), which also increases cGMP in the CNS
and periphery (Rutten et al., 2005), reversed memory deficits due to nNOS inhibition in
rats (Devan et al., 2006). In the present study, BAY-73-6691 was chosen because of its
high selectivity for PDE9. Also, PDE9 is distributed in brain regions involved in learning
and memory whereas PDE5 is expressed in the cerebellum and to a lesser degree the
hippocampus (Menniti et al., 2006).
The Km of PDE9 is in the range of 170nM for cGMP and the Vmax is about
4.9nM/min/μg of recombinant protein which is about twice as fast as the Vmax of PDE4
for cAMP (Fisher et al., 1998). This accelerated rate of cGMP hydrolysis may partially
explain why BAY-73-6691 was more successful at facilitating extinction than rolipram.
By blocking the catalytic activity of PDE9, the rapid increase in cGMP may have more
significant effects on downstream molecules that are involved in memory consolidation
(e.g. CREB). The impact of other PDEs that degrade cGMP may be less significant
because the pharmacokinetics of PDE9 may overshadow the kinetics of the other PDEs.
After considering PDE inhibitors that specifically increases cAMP (rolipram) or
cGMP (BAY-73-6691) we investigated whether a dual specificity PDE inhibitor which
elevates levels of both cAMP and cGMP would induce extinction of place preference.
Papaverine is a specific inhibitor of PDE10A that increased levels of cAMP and cGMP
(Siuciak et al., 2006) and improved phencyclidine-induced cognitive deficits in rats
(Rodefer et al., 2005). We found that acute injection of papaverine significantly elevated
cGMP levels in both the hippocampus and amygdala, but it only increased levels of
cAMP in the hippocampus. However, papaverine had no significant effect on extinction
of CPP (Fig. 3.2C).
Studies have shown that PDE10A is densely localized in the striatum but less so
in the hippocampus (Seeger et al., 2003), while PDE9 is highly localized in all sub-areas
of the hippocampus (van Staveren et al., 2002; 2004; Reyes-Irisarri et al., 2007). Given
the role of hippocampus in extinction learning, it is likely that inhibiting PDE9 has a
more profound effect on hippocampal functions than inhibiting PDE10A. This was
manifested through the behavioral experiment where BAY-73-6691 but not papaverine,
facilitated new learning and thereby induced extinction of cocaine-induced place
preference. Another possible explanation for the failure of papaverine to induce
extinction is that PDE9 metabolizes cGMP at a very high rate. Because of this, while
papaverine blocks hydrolysis of cGMP by PDE10A, PDE9 activity may increase and
quickly reduce levels of cGMP. In addition, PDE10A has a much higher affinity for
cAMP than cGMP (Boswell-Smith et al., 2006; Francis et al., 2010) and it does not
hydrolyze cGMP as efficiently as PDE9. This may also explain the finding that blockade
of PDE9 (BAY-73-6691) afforded extinction while blockade of PDE10A (papaverine)
did not (Fig. 3.2B, C). However, further studies are required to determine the long-term
effects of repeated administration of PDEi; this will help to elucidate whether a
compensatory action of PDEs could explain differential efficacies of the different PDEi.
In summary, the present study suggests that inhibitor of PDE9 has a prominent
role in the consolidation of extinction learning. It also appears that targeting a specific
PDE is more critical than targeting any PDE which metabolizes cGMP.

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