The pattern of cocaine conditioning differentially influences stress-induced reinstatement of conditioned place preference

Vulnerability to relapse following prolonged periods of abstinence presents a
major challenge to combating drug addiction. It is therefore essential that much attention
be placed on understanding the neural mechanisms that sub-serve relapse to drug use. We
have previously shown that the acquisition and reconsolidation of cocaine-context
memory associated with conditioning by a fixed daily dose (Fix-C) and escalating daily
doses (Esc-C) of cocaine engage different signaling pathways downstream of the Nmethyl-
D-aspartate receptor (NMDAR). The present study was undertaken to elucidate
the signaling molecules in stress-induced reinstatement of previously extinguished Fix-C
and Esc-C CPP in C57 BL6/J mice. Blockade of NMDAR signaling had differential
effects on forced swim-induced reinstatement of Fix-C and Esc-C CPP; MK-801
attenuated the reinstatement of Fix-C but not Esc-C CPP. Likewise, the neuronal nitric
oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI) attenuated stress-induced
reinstatement of Fix-C CPP but it had no effect on Esc-C CPP. The corticotrophin
releasing hormone receptor subtype 1 (CRH-R1) antagonist antalarmin also attenuated
stress-induced reinstatement of Fix-C but not Esc-C CPP. Antagonism of NR2Bcontaining
NMDARs had no effect on stress-induced reinstatement of neither Fix-C nor
Esc-C CPP. The suppressing effects of MK-801, 7-NI and antalarmin were not due to
inhibition of plasma stress hormone corticosterone levels. These results suggest
differential signaling pathways for stress-induced reinstatement of Fix-C and Esc-C CPP
where Fix-C CPP is CRH-R1-, NMDAR- and NO-dependent, while stress-induced
reinstatement of Esc-C CPP is likely mediated by different signaling molecules.
An important target for combating drug addiction is to understand the
neurobiological mechanisms that sub-serve relapse to drug use. Drug addiction is thought
to usurp the neural mechanisms of learning and memory (Hyman, 2005) and induce long
term plasticity as a result of changes in gene expression through chromatin remodeling
(McClung & Nestler, 2008). Memory of the rewarding effects of drugs of abuse is stored
in the brain even after prolonged periods of abstinence. This memory can resurface when
an individual is re-exposed to priming doses of the drug itself, drug-related cues or to
certain stressors (Shaham et al., 2003). To date, an effective treatment strategy for
preventing relapse to drug use following extended periods of abstinence remains a
Stress is an unavoidable part of life and it represents a major risk factor for
relapse susceptibility in abstaining addicts (Sinha, 2008). As such, understanding the
molecular factors that contribute to the re-establishment of drug seeking behavior
following exposure to stressful stimuli will aid in the development of effective
pharmacological therapies for the treatment of drug addiction. The physiological
response to stress involves the activation of the hypothalamic-pituitary-adrenal (HPA)
axis (Harbuz & Lightman, 1992). In response to stressful stimuli, corticotrophin-releasing
hormone (CRH) is secreted by the hypothalamus. CRH stimulates the release of
adrenocorticotrophic hormone (ACTH) from the anterior pituitary which subsequently
induces the release of corticosteroids from the adrenal cortex (Knol, 2011).
Corticosteroids such as glucocorticoids play an important role in metabolism, immune
response and behavior (Sapolsky et al., 2000) as well as in regulation of the HPA axis
through negative feedback (Dallman, 2005). Along with the HPA axis, the extrahypothalamic
CRH system is also activated in response to stressful stimuli. Indeed, the
CRH receptors subtype 1 (CRH-R1) of extra-hypothalamic CRH system has been shown
to mediate stress-induced changes in cocaine reward (Goeders, 2002).
The conditioned place preference (CPP) paradigm, which is based on the
principles of Pavlovian conditioning, is used to model different features of addictive
behavior (Sanchis-Segura & Spanagel, 2006). Traditionally, in animal models of stressinduced
reinstatement in self-administration studies, the stressor (e.g., footshock) must be
given in the same context as the conditioning drug (Shalev et al., 2000). However,
stressors outside the drug-taking environment are able to precipitate relapse in humans
(Shiffman, 1982). Studies have shown that stressors such as mild foot-shock and the
forced swimming test (FST) are capable of reinstating previously extinguished place
preference for a variety of drugs of abuse including cocaine (Wang et al., 2000),
morphine (Ma et al., 2007) and alcohol (Bhutada et al., 2012). Additionally, the CRH
receptor has been shown to play a critical role in the effects of stress on reinstatement of
CPP (McReynolds et al., 2014).
We have previously shown that cocaine-associated memory acquired through
different schedules of cocaine administration show differences in the magnitude and
persistence of CPP. Mice conditioned by escalating daily doses of cocaine (Esc-C)
display higher magnitudes of CPP and were resistant to extinction by non-reinforced
exposure to the training context compared to mice conditioned by fixed daily doses of
cocaine (Fix-C) where CPP is readily extinguished (Itzhak & Anderson, 2012).
Furthermore, we found that mice conditioned by Esc-C showed higher levels of NR2Bcontaining
NMDARs compared to Fix-C. Additionally, while antagonism of NR2Bcontaining
NMDARs was equally effective at attenuating the acquisition and disrupting
the reconsolidation of both Fix-C and Esc-C memory, inhibition of nNOS signaling was
effective against Fix-C but not Esc-C memory (Liddie & Itzhak, 2014, Chapter 2). Given
these observations, our goal was to determine whether differences observed in the
acquisition and extinction of CPP between Fix-C and Esc-C would also hold true when
assessing stress-induced reinstatement. The present study was undertaken to elucidate the
contribution of different signaling pathways to the re-establishment of previously
extinguished Fix-C and Esc-C CPP evoked by exposure to a stressful stimulus, the forced
swim test (FST).

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