The effect of phosphodiesterase inhibitors on the extinction of cocaine-induced conditioned place preference in mice

everal phosphodiesterase inhibitors (PDEi) improve cognition, suggesting that
an increase in brain cAMP and cGMP facilitates learning and memory. Since extinction
of drug seeking behavior requires associative learning, consolidation and formation of
new memory, the present study investigated the efficacy of three different PDEi in
extinction of cocaine-induced conditioned place preference (CPP) in B6129S mice. Mice
were conditioned by escalating doses of cocaine which was resistant to extinction by free
exploration. Immediately following each extinction session mice received a)
saline/vehicle, b) rolipram (PDE4 inhibitor), c) BAY-73-6691 (PDE9 inhibitor) or d)
papaverine (PDE10A inhibitor). Mice that received saline/vehicle during extinction
training showed no reduction in CPP for >10 days. BAY-73-6691 a) dose-dependently
increased cGMP in hippocampus and amygdala b) significantly facilitated extinction and
c) diminished the reinstatement of cocaine CPP. Rolipram, which selectively increased
brain cAMP levels, and papaverine which caused increases in both cAMP and cGMP
levels, had no significant effect on extinction of cocaine CPP. Results suggest that
increase in hippocampal and amygdalar cGMP levels via blockade of PDE9 has a
prominent role in the consolidation of extinction learning.
The administration of a drug that changes the affective state of the organism in a
specific context triggers an associative learning process and the formation of long-term
memory (LTM). The expression of conditioned place preference (CPP) is viewed as a test
for reactivity to drug-associated conditioned stimulus (CS); this test has validity for cuereactivity
in human drug users. The CPP paradigm has also been used to investigate
extinction of “drug-seeking behavior” and reinstatement of conditioned response (Aguilar
et al., 2009; Itzhak & Martin, 2002; Parker & McDonald, 2000; Mueller & Stewart,
2000; Mueller et al., 2002). Interestingly, reinstatement of cocaine CPP following
extinction is a drug-specific phenomenon that can be triggered only by drugs that share a
similar mechanism of action with that of cocaine (Itzhak & Martin, 2002). Therefore, the
reinstatement of place preference, like the reinstatement of drug self-administration
presents a meaningful resource for the study of relapse. Animal and human studies
suggest that re-exposure to a low dose of psychostimulants, opiates or alcohol, following
abstinence or extinction of drug use, may cause relapse (Shaham et al., 2003). It is
therefore critical to develop pharmacotherapies and behavioral practices by which
extinction of drug seeking behavior will ultimately result in resistance to both drugassociated
cues and drug-priming.
Extinction learning by “exposure therapy” is thought to be essential for the
management of drug addiction (Carter & Tiffany, 1999; Powell et al., 1993; Siegel &
Ramos, 2002). Extinction typically requires long or multiple re-exposures to a CS
(Nader, 2003; Power et al., 2006). Results from fear conditioning studies suggest that the
extinction process does not eliminate or cause ‘unlearning’ of the initial conditioned
response; rather, the organism learns that the CS no longer elicits the previous stimulus
(Bouton, 2002; 2004; Havermans & Jansens, 2003). Thus, extinction requires associative
learning, consolidation and formation of new memory (Milad & Quirk, 2002; Santini et
al., 2001).
Through the activation of their respective kinases (PKA and PKG), cyclic
nucleotide (cAMP and cGMP) signaling pathways are important regulators of neural
function and synaptic homeostasis (Bales et al., 2010). While adenylyl cyclase (AC) and
guanylyl cyclase (GC) generate the second messengers cAMP and cGMP respectively,
phosphodiesterases (PDEs) hydrolyze these cyclic nucleotides into their inactive
monophosphates, 5’-AMP and 5’-GMP, and thereby contribute to the regulation of their
intracellular levels (Essayan, 2001). Eleven different families of mammalian PDE’s have
been identified in the CNS and periphery. All neurons express multiple PDEs which
differ in cyclic nucleotide specificity, affinity, regulatory control and subcellular
distribution (Bender & Beavo, 2006; Blokland et al., 2006; Boswell-Smith et al., 2006;
Menniti et al., 2006).
The differential localization of PDEs in the CNS and periphery determine how
effective phosphodiesterase inhibitors (PDEi) are at regulating different processes. Brain
PDEs include PDE1, PDE2, PDE4, PDE5, PDE9, PDE10 and PDE11. PDE4 is widely
distributed throughout the brain (Bender & Beavo, 2006). The PDE4 inhibitor rolipram
has been used as an anti-inflammatory and has shown antipsychotic-like therapeutic
effects (Kelly et al., 2007). Rolipram enhances learning and memory in various
paradigms (Cheng et al., 2010; Monti et al., 2006; Rose et al., 2005; Tully et al., 2003;
Zhang & O’Donnell, 2000), but unexpectedly it disturbed expression and extinction of
conditioned fear in mice (Mueller et al., 2010). PDE9 is highly localized in all sub-areas
of the hippocampus (van Staveren et al., 2002; 2004; Reyes-Irisarri et al., 2007) and the
specific PDE9 inhibitor BAY-73-6691 improves learning and memory in rodents (van
der Staay et al., 2008). PDE10A is densely localized in the striatum but less in the
hippocampus (Seeger et al., 2003). Papaverine is a specific inhibitor of PDE10A that
increased levels of cAMP and cGMP (Siuciak et al., 2006) and improved phencyclidineinduced
cognitive deficits in rats (Rodefer et al., 2005).
The use of selective PDEi as potential cognitive enhancers is suggested by studies
in which PDEi facilitated learning and memory in animal models with experimentally
induced learning and memory deficits (Bender & Beavo, 2006; Blokland et al., 2006;
Boswell-Smith et al., 2006; Menniti et al., 2006). However, it is unclear whether
selective PDEi facilitate learning and memory in subjects with no cognitive impairments.
Therefore, the effects of the PDEi on extinction learning may be different than their
effects on improving learning following cognitive deficits.
The hippocampus and amygdala are implicated in spatial/contextual and
emotional/cued memory, respectively. We hypothesized that increases in hippocampal
and amygdalar cyclic nucleotide levels through the action of PDE inhibitors will facilitate
extinction learning of cocaine-induced place preference. We sought to investigate PDE
inhibitors with different specificities to cAMP and cGMP in order to determine which if
any group of PDE inhibitors has a more prominent role in the consolidation of extinction
learning. We first determined how cyclic nucleotide levels were affected in the
hippocampus and amygdala in response to a PDE4 (cAMP specific) inhibitor rolipram, a
PDE9 (cGMP specific) inhibitor BAY-73-6691 and a PDE10A (dual specificity) inhibitor
papaverine. Then, the efficacies of these PDE inhibitors to extinguish and prevent
reinstatement of cocaine CPP were investigated. We report that of the three PDE
inhibitors only BAY-73-6691, which increased hippocampal and amygdalar cGMP
levels, induced the extinction and attenuated the reinstatement of cocaine place

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