Activation and role of the medial prefrontal cortex (mPFC) in extinction of ethanol-induced associative learning in mice

Abstract

Although the medial prefrontal cortex (mPFC) has been shown to be integrally involved in extinction of a number of associative behaviors, its role in extinction of alcohol (ethanol)-induced associative learning has received little attention. Previous reports have provided evidence supporting a role for the mPFC in acquisition and extinction of amphetamine-induced conditioned place preference (CPP) in rats, however, it remains unknown if this region is necessary for extinction of ethanol (EtOH)-induced associative learning in mice. Using immunohistochemical analysis of phosphorylated and unphosphorylated cAMP response element-binding protein (CREB), the current set of experiments first showed that the prelimbic (PL) and infralimbic (IL) subregions of the mPFC exhibited dynamic responses in phosphorylation of CREB to a Pavlovian-conditioned, EtOH-paired cue. Interestingly, CREB phosphorylation within these regions was sensitive to manipulations of the EtOH-cue contingency—that is, the cue-induced increase of pCREB in both the PL and IL was absent following extinction. In order to confirm a functional role of the mPFC in regulating the extinction process, we then showed that electrolytic lesions of the mPFC following acquisition blocked subsequent extinction of EtOH-CPP. Together, these experiments indicate a role for the PL and IL subregions of the mPFC in processing changes of the EtOH-cue contingency, as well as in regulating extinction of EtOH-induced associative learning in mice.

Introduction

Rehabilitation from drug- and alcohol-associated addictive behaviors represents a uniquely difficult task in the clinic. Because patients who pursue rehabilitation treatments have most often spent many years with an addiction, breaking the control that these drug-seeking behaviors exert over a patient’s everyday life is a daunting task—one that most often ends in relapse. Development of, and relapse to, addictive disorders is thought to be strongly influenced by a broad spectrum of drug-associated environmental cues and contexts that, following years of drug-related learning, are able to initiate and maintain drug-seeking behaviors (for review see Duka, Crombag, & Stephens, 2010). It is for this reason that rehabilitation treatments of drug- and alcohol-associated addictive disorders have included cue-exposure therapy (CET)—a type of extinction training during which previously drug-paired cues are presented in a nonreinforced manner in hopes of reducing their valence and subsequent behavioral control (e.g., Drummond & Glautier, 1994). However, despite the effectiveness of extinction to reduce drug-seeking behavior in animals (for review see Millan, Marchant, & McNally, 2011), cue-exposure therapy has been largely ineffective at increasing rates of abstinence (Conklin & Tiffany, 2002). Therefore, it is important to continue to further our understanding of the neurobiological substrates underlying extinction of drug- and alcohol-seeking behavior in animals in order to improve rehabilitation treatments, including CET, in humans.

In the conditioned place preference (CPP) model of drug-induced associative learning, a neutral stimulus attains significant incentive value after being repeatedly paired with a drug of abuse such as ethanol (EtOH) (Cunningham, Groblewski, & Voorhees, 2011). Following this straightforward Pavlovian-conditioning procedure, approach behavior to the EtOH-paired conditioned stimulus (CS) is assessed during a preference test. In subsequent extinction of CPP, animals are exposed to the same set of cues, but in a nonreinforced manner (i.e., in the absence of EtOH). Similar to acquisition, extinction of CPP represents associative learning that depends on reward-prediction error, and both phases of learning can be represented by negative exponential functions of associative strength, as reflected in the model described by Rescorla and Wagner (1972). The extinction phase of associative behaviors such as CPP is thought to weaken the conditioned response elicited by the CS through an inhibitory mechanism (Konorski, 1967, Pavlov, 1927). Furthermore, the behavioral inhibition observed during extinction is thought to involve de novo memory formation that leaves the original association intact (Bouton, 2004).

Recent evidence suggests that the medial prefrontal cortex (mPFC) is involved in both the expression and extinction of conditioned fear and operant drug-seeking behavior (for review see Peters, Kalivas, & Quirk, 2009). Specifically, the prelimbic (PL) subregion has been suggested to be primarily involved in expression of conditioned behaviors while the infralimbic (IL) subregion is involved in consolidation (and therefore also retrieval) of extinction (Quirk and Mueller, 2008, Vidal-Gonzalez et al., 2006). Following extinction, exposure to cues capable of reinstating drug-seeking behavior has been shown to cause significant activation of both the PL and IL subregions of the mPFC (Zavala, Biswas, Harlan, & Neisewander, 2007). With regards to expression of non-operant drug-induced associative learning, a brief cocaine-CPP expression test resulted in significant activation of the PL (Miller & Marshall, 2005) while inactivation of the mPFC (both PL & IL) impaired extinction of amphetamine-CPP (Hsu & Packard, 2008). Additionally, inactivation of the mPFC reinstated heroin-CPP, an effect the authors attributed to a disinhibition of place preference expression (Ovari & Leri, 2008). Contrary to these findings however, Zavala, Weber, Rice, Alleweireldt, and Neisewander (2003) reported that PL lesions had no effect on acquisition, expression, or extinction of cocaine-induced CPP. Thus, despite its broad support for a role in extinction of conditioned fear and drug-induced operant behavior, there remains conflicting evidence for the role of the mPFC in extinction of CPP.

The involvement of the mPFC in extinction of ethanol (EtOH)-induced associative learning has received even less attention and it remains unknown if the PL and IL subregions within the mPFC are necessary for extinction of Pavlovian-conditioned EtOH-seeking behaviors such as EtOH-CPP in mice. Previous reports that involved operant EtOH procedures have shown inconsistent evidence for the involvement of mPFC in extinction of EtOH-seeking behavior. Specifically, Dayas, Liu, Simms, and Weiss (2007) reported cue-induced activation of the mPFC following exposure to cues capable of reinstating self-administration in rats after extinction. In contrast, however, Hamlin, Newby, and McNally (2007) reported no significant effect of extinction on mPFC activation after contextual renewal of EtOH-responding. Interestingly, Topple, Hunt, and McGregor (1998) showed significant PL activation following exposure to the EtOH-paired operandum during an extinction session. Thus, although the data remain somewhat inconsistent, it appears that the mPFC may be activated in situations when EtOH is expected but not received (i.e., a large EtOH-reward prediction error exists), such as extinction and/or reinstatement. However, the aforementioned experiments were performed using operant procedures in rats and thus it remains unknown how extinction of the EtOH-cue contingency would affect mPFC activation elicited by a Pavlovian-conditioned cue in the absence of an instrumental response.

Therefore, although the literature supports the assertion that the PL and IL subregions exhibit differential roles in expression and extinction of cue-induced behaviors (e.g., Peters et al., 2009), these studies have primarily been performed in operant procedures in rats with drugs other than EtOH, and it is therefore unclear if the distinction between these subregions generalizes across species and to extinction of EtOH-induced associative learning in mice.

The current experiments were performed in order to first understand how extinction of an EtOH-cue association affects cue-induced activation of the mPFC and second, to examine the effects of direct manipulations of the mPFC on extinction of EtOH-induced associative learning in mice. Experiment 1 utilized immunohistochemical (IHC) analysis of the activated transcription factor, phospho-cAMP response element-binding protein (pCREB), to examine the effects of extinction on mPFC (PL and IL) activation following a brief exposure to an EtOH-paired cue capable of eliciting approach behavior in mice. Experiment 2 then used electrolytic lesions of the mPFC to confirm a functional role of this region in extinction of EtOH-CPP in mice. It was hypothesized that: (1) the mPFC would show cue-induced expression of pCREB that was sensitive to changes in the EtOH-cue contingency following extinction, and (2) mPFC lesions would impair extinction of EtOH-CPP. These experiments were intended to further clarify the roles of the PL and IL subregions of the mPFC in processing changes of EtOH-cue contingencies that underlie extinction of EtOH-induced associative learning in mice.