The Nucleus Accumbens: Mechanisms of Addiction across Drug Classes Reflect the Importance of Glutamate Homeostasis

A. What Are We Trying to Model in Experimental Animals?

Animal models are the most efficient method for determining how gene expression and cell signaling in specific brain circuits mediate learning and memory as well as the expression of motivated behavior based on learned associations. However, when we are extrapolating to a behavioral disorder that is defined in part by uniquely human criteria, it is important to accept the limitations of animal models. Models of addiction have evolved over the last 2 decades to provide increasing face validity through anthropomorphizing rodent behavior (Piazza and Deroche-Gamonet, 2014) but have been less successful at producing predictive validity in terms of drug development. However, the use of anthropomorphic models of addiction has produced procedures that yield behaviors that appear similar to certain human addiction endophenotypes, such as impulsivity, escalating drug use, intrusive thinking, or compulsive drug seeking (Ahmed et al., 2002; Everitt et al., 2008; Perry and Carroll, 2008; Dalley et al., 2011; Chen et al., 2013). However, below we argue that there are limits in extrapolating from rodent to human behavior and that modeling shared neurobiological processes that are similarly altered by addictive drugs may be the most useful approach.

To encapsulate both glutamatergic physiology and the relevance of various animal models of addiction, below we focus on models that have successfully revealed involvement of the circuitry providing glutamatergic synapses to the NAc. In particular, we focus on the innervations arising from cortical regions, such as the prelimbic cortex (PLC) and infralimbic cortex (IFC) in rodents and the anterior cingulate and subgenual PFC in humans, and allocortical regions, such as the basolateral amygdala (BLA) and ventral hippocampus in both humans and rodents (Fig. 1). As indicated above, the focus on these synapses arises from their role in a defining characteristic of addiction—namely, a failure to suppress the overwhelming motivation to relapse to drug use.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1.

NAc connectivity. The NAc receives inputs from cortical, allocortical, thalamic, midbrain, and brainstem structures. In turn, it sends projections to other basal ganglia nuclei (VP and substantia nigra pars reticulata), nuclei in the mesencephalon, the hypothalamus, and the extended amygdala. Note that many structures project from different subareas to the NAcore or NAshell. For clarity, these projections have been color coded as projecting to the NAcore (green), medial NAshell (light blue), or lateral NAshell (dark blue); in reality, many regions project to both the NAcore and NAshell along topographical gradients (e.g., dorsoventral projections from the hippocampus terminating from lateral to medial parts of the accumbens; shown as color gradients in the figure). A number of regions project uniformly throughout the accumbens and are marked white. A8, retrorubral area; ACC, anterior cingulate cortex; AId, dorsal anterior insular; AIv, ventral anterior insular; dHPC, dorsal hippocampus; dlVP, dorsolateral ventral pallidum; DRN, dorsal raphe nucleus; IL, infralimbic cortex; ILT, interlaminar nuclei of the thalamus; LC, locus coeruleus; LH, lateral hypothalamus; LPO, lateral preoptic area; NTS, nucleus of the solitary tract; PL, prelimbic cortex; PPN, pedunculopontine nucleus; PVT, paraventricular nucleus of the thalamus; vlVP, ventrolateral ventral pallidum; vmVP, ventromedial ventral pallidum; SNc, substantia nigra pars compacta; SNpr, substantia nigra pars reticulata.

B. Using Animal Models to Understand Constitutive and Transient Adaptations

In applying animal models of addiction toward understanding the neurobiology of the addicted state, investigators are asking two fundamental questions: 1) What are the long-lasting, constitutive changes produced by addictive drugs that may constitute the addiction? 2) What are the transient neurologic processes that mediate the expression of behavioral pathology? Although animal models are behaviorally defined by the expression of the behavioral effects of a drug (e.g., behavioral sensitization or drug seeking), the neurobiological analyses are largely made after a period of withdrawal or abstinence. In other words, animal models have focused on the second question in terms of behavior and the first question in terms of understanding addiction neurobiology. We will reflect on this disconnect as we proceed to analyze each model in terms of value in answering both questions.

C. Motor Sensitization

Sensitization occurs when repeated exposure to a stimulus augments a behavioral or physiologic response compared with the first stimulus presentation. In terms of drugs of abuse, cocaine, amphetamines, nicotine, ethanol, morphine, and Δ-9-tetrahydrocannabinol (THC) have all been shown to produce sensitization of locomotor behavior in animal models (Vezina and Stewart, 1989; Borowsky and Kuhn, 1991; Paulson and Robinson, 1995; Cadoni and Di Chiara, 2000; Cadoni et al., 2001; Quadros et al., 2002). Sensitization is most commonly assayed by measuring increases in locomotor activity after repeated experimenter-administered (noncontingent) drug delivery. Although a single drug injection may be sufficient to elicit behavioral sensitization lasting a few days (Post and Weiss, 1988; Valjent et al., 2010), repeated drug administration more generally appears necessary to produce enduring (weeks to months) sensitization. In rodent models of sensitization, augmented behavior to an acute drug challenge is reliably paralleled by enhanced dopaminergic activity in the NAc (Kalivas and Duffy, 1990; Johnson and Glick, 1993; Paulson and Robinson, 1995; Cadoni and Di Chiara, 2000) (with the possible exception of alcohol; see Zapata et al., 2006). In contrast with dopamine, repeated noncontingent drug exposure variably decreases basal levels of extracellular glutamate in the NAc in the case of cocaine or has no effect in the case of amphetamine (Pierce et al., 1996; Xue et al., 1996). In addition, when behavioral sensitization is expressed by a subsequent noncontingent drug injection, there is an increase in nucleus accumbens core (NAcore) extracellular glutamate that depends on the presence of environmental cues associated with previous drug exposure and occurs only in rats that actually show a sensitized behavioral response (Bell et al., 2000; Hotsenpiller et al., 2001). This linkage between glutamate release and learned drug–environment associations is manifested less with dopamine, in which the extracellular levels are elevated as part of the acute pharmacological action of the drug, although drug–environment associations can augment the drug-induced increase in extracellular dopamine (Badiani et al., 1995; Bell et al., 2000). Importantly, in humans and nonhuman primates, elevated dopamine appears to be more dependent on the presence of a drug-associated context or cues (Bradberry, 2007; Narendran and Martinez, 2008; Vezina and Leyton, 2009).

In parallel with the basal levels of extracellular glutamate being variably altered by a behavioral sensitizing treatment protocol depending on the addictive drug, glutamate receptor (GluR) levels in the accumbens also vary depending on the drug being used. Notably, whereas repeated cocaine administration elicits a time-dependent increase in surface expression of the GluR1 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors that is present after at least 1 week but not on day 1 of withdrawal, a sensitizing treatment regimen of morphine or amphetamine does not consistently alter the level of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) or N-methyl-d-aspartic acid receptor (NMDAR) subunits (Boudreau and Wolf, 2005; Boudreau et al., 2007; Kourrich et al., 2007; Ghasemzadeh et al., 2009; Ferrario et al., 2011). The changes produced by repeated cocaine on GluR1 levels are paralleled by enduring increases in AMPA currents, which are quantified as the AMPA/N-methyl-d-aspartic acid (NMDA) ratio and the density of dendritic spines in accumbens medium spiny neurons (MSNs) (Thomas et al., 2001, 2008; Norrholm et al., 2003; Robinson and Kolb, 2004; Kourrich et al., 2007; Shen et al., 2009; Dietz et al., 2012). However, the increase in spine density does not occur after daily noncontingent administration of morphine (Robinson and Kolb, 1999). The morphologic component of drug-induced plasticity is discussed in greater detail in section V below.

In summary, although substantial face validity of behavioral sensitization is lost because the drug is experimenter administered, the administration protocol induces some forms of plasticity at glutamatergic synapses. However, the most replicable effects of noncontingent drug administration in the sensitization model, regardless of the addictive drug, are on dopamine neurons in the ventral tegmental area (VTA) and in releasing dopamine in the NAc (Kalivas and Stewart, 1991; Jones and Bonci, 2005). Thus, many investigators have used a variety of addictive drugs and stress to show that the initiation (development) of sensitization by repeated drug injection depends on adaptations in excitatory and peptidergic afferents to the VTA and transient changes in glutamate synaptic strength on dopamine neurons (Bonci and Borgland, 2009; Lüscher and Malenka, 2011). In addition, the majority of studies show that the expression of locomotor sensitization is associated with sensitized release of dopamine in the accumbens (Steketee and Kalivas, 2011). In contrast with dopamine, behavioral sensitization is not as consistently associated with drug-induced changes in glutamate transmission in the accumbens, as discussed above. Not only do different classes of drugs producing behavioral sensitization elicit distinct enduring changes in AMPARs and spine morphology in accumbens MSNs, but there is also a requirement for contextual associations with the drug in developing and expressing glutamate release and synaptic plasticity. Although the expression of sensitized drug-induced locomotion can be conditioned to and made dependent on contextual cues (Stewart, 1991; Crombag et al., 2000), it is also clear that behavioral sensitization can be induced without associating the unconditioned motor response with a conditioning stimulus or context. This is perhaps most clearly demonstrated by the fact that intra-VTA injections of amphetamine do not elicit an unconditioned locomotor response, whereas they do induce enduring locomotor sensitization to a subsequent systemic or intra-accumbens injection of amphetamine or cocaine (Kalivas and Weber, 1988; Vezina, 1993). Taken together, these data indicate that although drug-induced dopamine release sensitizes in parallel with behavior, it is necessary to develop learned associations between the unconditioned drug response and contextual or discrete environmental cues in order to engage the cortical and allocortical inputs to the accumbens (Fig. 1) with noncontingent injections. Unfortunately, most studies have not carefully controlled learned associations made with noncontingent drug effects, resulting in the sensitization literature identifying variable levels of behavior and glutamatergic adaptations. Another important confound of sensitization, with the exception of a few studies regarding contextual cues (Badiani et al., 1995; Uslaner et al., 2001), is that the majority of studies on the expression of sensitization rely on an acute drug injection to elicit the behavior. Accordingly, the distinct acute pharmacology of each class of drug can produce reversible changes that may confound or mask measures of glutamate transmission contributing to the sensitized behavioral response. In addition to the confounding acute effects of the drug, the face validity of the sensitization model is also limited by the fact that a sensitized dopamine response is largely absent in monkeys and humans with high levels of cumulative exposure (Bradberry, 2007).

D. Conditioned Place Preference

The marked and variable effect of learned associations on behavioral and cellular measures in the behavioral sensitization paradigm is better controlled in the conditioned place preference (CPP) protocol (Tzschentke, 2007). CPP is a simple form of classic conditioning or Pavlovian conditioning, a learning process that involves either positive or negative associations between two stimuli. In either case, a conditioned stimulus (CS) or a previously neutral stimulus that does not elicit a response gains predictive value over the occurrence of an unconditioned stimulus (US) (e.g., acute experimenter-delivered drug injection) through training. The CPP procedure generally consists of three phases: habituation, conditioning, and testing. During habituation, the animal is allowed to move freely throughout a test apparatus that is most often of a two-chamber or three-chamber construction. At this time, initial preference is measured and the researcher may assign treatment pairings in a biased or unbiased design, a choice that can affect the final results. In an unbiased design, subjects are randomly assigned regardless of their initial preferences. In a biased design, the initially nonpreferred side is paired with the test drug. During conditioning, one chamber of the apparatus is paired with the drug, whereas the other side is paired with vehicle injection. This training involves multiple pairings of each contextually distinct compartment with the drug or vehicle over a period of several days, but protocols vary in the number and schedule of pairings. After training, preference is tested in a drug-free state by measuring the amount of time spent in each chamber. The choice of one context over the other is said to impart information regarding the drug-induced motivational state. If the drug is “rewarding,” the subject is expected to spend more time in the drug-paired environment, thus producing CPP (Bardo and Bevins, 2000). Conversely, if the drug induces a negative state, the subject will avoid the paired context, producing a place aversion (Mucha et al., 1982).

Under the correct conditions, cocaine, amphetamines, ethanol, and morphine have all been shown to produce a CPP (Tzschentke, 2007). Recently, place preference for amphetamines was demonstrated in humans (Childs and de Wit, 2009). Early research suggested an involvement of D1 dopamine receptors and NMDA-type glutamate receptors in the establishment of cocaine CPP, whereas the AMPA-type glutamate receptors seem to be involved in CPP expression as elucidated by using systemic delivery of specific AMPA and NMDA inhibitors (Cervo and Samanin, 1995). However, either the AMPA/kainate antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) or the D1/D2 dopamine antagonist fluphenazine delivered directly to the accumbens reduces CPP expression, whereas only DNQX affects acquisition in rats trained with cocaine (Kaddis et al., 1995). Similarly, methamphetamine CPP is attenuated by intracerebroventricular pretreatment with the antagonist at GluN2B containing NMDA channels ifenprodil, DNQX, or the metabotropic glutamate receptor (mGluR) 5 negative allosteric modulator 2-methyl-6-(phenylethynyl)pyridine (MPEP) during pairing (Miyatake et al., 2005). These data indicate that glutamatergic activity may be crucial for learning the association between environmental stimuli and drug reward and identify a locus of action in the mesoaccumbens pathway (see section VI for more detailed pharmacology of glutamate receptors in CPP).

CPP can be also be extinguished, and reinstatement can be measured as a proxy of drug craving and relapse (Mueller and Stewart, 2000). Extinction is facilitated by repeatedly administering vehicle injections in both compartments or conducting repeated preference testing until preference is extinguished. Extinguished CPP can be reinstated with a drug-priming injection (or in some cases, stress). A role for glutamatergic transmission has been demonstrated in CPP reinstatement. For instance, NMDAR antagonists have been used to suppress cocaine- and morphine-primed reinstatement of place preference (Ribeiro Do Couto et al., 2005; Maldonado et al., 2007). Many of the results obtained using the CPP reinstatement procedure are complementary with self-administration studies (see below), but these models evaluate different aspects of reward (conditioned approach versus operant responding); thus, the findings are not always consistent. For example, although memantine (an NMDA antagonist) reduced reinstatement of cocaine CPP, it did not block cocaine-primed reinstatement in the operant task. The drug did, however, abolish lever discrimination by increasing inactive lever responses (Bespalov et al., 2000; Maldonado et al., 2007).

The CPP paradigm is a popular drug-screening tool in animal models because it is an inexpensive and efficient procedure. Moreover, with the addition of a reinstatement test in many studies, the CPP paradigm is useful to evaluate both the development and expression of drug-induced behavioral and neurologic adaptations. However, reinstating CPP is usually accomplished by acute readministration of the drug since the conditioned context has been extinguished and, as discussed above, the acute drug pharmacology may interfere with the fidelity of measures of glutamatergic transmission relevant to addiction. From the perspective of engaging cortical and allocortical inputs to the NAc, the conditioned associations activated in CPP likely lead to activation of this circuit in the process of recalling learned information to guide behavior. Although this has not been directly evaluated at the level of extracellular glutamate levels, daily noncontingent cocaine injections using a CPP protocol produce enduring increases in dendritic spine density in accumbens MSNs (Pulipparacharuvil et al., 2008; see the discussion on morphologic plasticity in section V). In conclusion, although it involves noncontingent drug administration, this protocol is appropriate for consistently engaging cortical and allocortical afferents to the accumbens because of the requirement for a learned contextual association to express CPP. Because the expression of context-induced place preference is drug free, it is possible to perform studies quantifying changes in glutamatergic plasticity or transmission initiated by the drug-paired context. As an example, changes in synaptic AMPAR expression were observed in the hippocampus when animals were re-exposed to a morphine-paired context, even if they received a saline injection (Xia et al., 2011). Furthermore, morphine CPP also increased basal synaptic transmission, altered synaptic levels of NMDAR subunits, and inhibited hippocampal long-term potentiation (LTP) (Portugal et al., 2014). Although the CPP protocol has drawbacks in terms of requiring drug-induced reinstatement, it is useful for determining the neural plasticity associated with drug-induced learned behavior. Indeed, in transgenic mouse models in which establishing self-administration is technically challenging, CPP has been the choice of noncontingent drug treatment paradigms to evaluate addiction-associated neuroadaptations (Russo et al., 2010). However, even using transgenic mice, the literature is gradually moving from CPP to self-administration models of addiction because of the latter’s greater face validity with human addiction. It is also unclear whether CPP is isomorphic with drug self-administration, because some drug classes elicit one drug-related behavior but not the other (Bardo and Bevins, 2000).

E. Self-Administration

More complex animal models of drug addiction are based on the analysis of behavioral output using schedules of reinforcement, established by Ferster and Skinner (1957). Instrumental behavior occurs because it was previously involved in producing certain consequences (Weeks, 1962; Schuster and Thompson, 1969; Domjan, 2003). Modern approaches to studying instrumental conditioning in drug addiction include operant responses (e.g., a lever press or nose poke on an opperandum) that lead to the delivery of an US (e.g., an intravenous drug infusion). This procedure of positive reinforcement is termed self-administration. Self-administration is frequently used to model addiction because it more closely resembles the human condition compared with an experimenter-delivered drug. In self-administration models, animals are placed in operant chambers, and completion of a schedule of reinforcement via lever presses or nose pokes is accompanied by intravenous or oral drug delivery. Usually, fixed-ratio (FR) schedules are used in self-administration models, such that an animal is required to press a lever a fixed number of times prior to drug delivery. Alternatively, progressive-ratio (PR) schedules are used to examine the reinforcing efficacy of a drug (or the probability that a drug will serve as a reinforcer). In a PR schedule, an animal must produce an increasing number of responses on an opperandum for each successive reinforcer. The self-administration model can be used to model various components of human drug use, including learning to take the drug (acquisition), stable regular drug use (maintenance), progressively increasing and compulsive drug use (escalation), drug abstinence (withdrawal with or without extinction of responding for the drug), and relapse to drug seeking (reinstated or context-induced responding).

A. Medium Spiny Neurons

The principle cell type in the striatum is the GABAergic MSN, which comprises approximately 90%–95% of the total neuronal population (Fig. 2). These cells can be subdivided into two distinct subpopulations based on characteristic dopamine receptor (D1/D2) and neuropeptide expression profiles (Gerfen and Surmeier, 2011). D1 receptor–containing MSNs coexpress dynorphin, substance P, and M4 cholinergic receptors, whereas D2 MSNs express enkephalin, neurotensin, and A2a adenosine receptors (Le Moine and Bloch, 1995; Lobo et al., 2006). Dopamine receptors on MSNs are G protein–coupled receptors with largely opposing effects on intracellular signaling cascades, leading to differential responses to dopamine and imbuing the separate cell populations with distinct physiologic properties. D1-type dopamine receptors are coupled to the Gα_s/olf family of G proteins that activate adenylyl cyclase to stimulate cAMP production and activation of downstream signaling cascades via cAMP-dependent protein kinase and other cAMP-dependent proteins, which ultimately regulate gene expression via transcription factors including cAMP response element binding protein (CREB) (Beaulieu and Gainetdinov, 2011). The D2-type dopamine receptors couple to Gα_i/o proteins that inhibit adenylyl cyclase and cAMP production, resulting in directly opposing effects on intracellular signaling and gene expression (Beaulieu and Gainetdinov, 2011). It has long been appreciated that these two populations display unique biochemical properties based on differences in dopaminergic signaling and gene expression profiles. The development of D1- and D2-fluorescent coupled bacterial artificial chromosome (BAC) transgenic mice, along with other technical advancements, allows investigators to more thoroughly explore the differences between D1- and D2-expressing MSNs both at a basic level and in disease models (Matamales et al., 2009; Valjent et al., 2009).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

NAc: the usual suspects. A general schematic of the some of the cell types discussed in this review that are present in the NAc, including MSNs (light blue), astrocytes (yellow), and various types of interneurons (purple). The accumbens receives inputs from several brain regions; examples of neurons that synapse in the accumbens are glutamatergic projection neurons (green) as well as dopaminergic projection neurons (red) (for more detail see Fig. 2).

Regarding MSNs and drug-related behaviors, using BAC reporter strains reveals that both populations of cells make differential contributions to drug-associated behaviors, and drug-induced alterations in structure and function vary in the two subpopulations (Gong et al., 2003). Noncontingent cocaine injections induce phosphorylation of protein kinase A (PKA), extracellular signal–regulated kinase (ERK), and histone H3 specifically in D1 MSNs, whereas they reduce phospho-PKA and phospho-ERK in D2 MSNs (Bertran-Gonzalez et al., 2008; Goto et al., 2015). A recent comprehensive study examined the induction of ΔFosB in response to cocaine, ethanol, THC, and μ-opiates in D1 and D2 MSNs throughout the striatum and described drug-specific patterns of induction (Lobo et al., 2013). For example, cocaine, ethanol, and THC induced ΔFosB expression only in D1 MSNs in the NAcore, NAshell, and dorsal striatum, whereas morphine and heroin significantly induced ΔFosB in both cell types. Interestingly, similar patterns were observed between experimenter-administered and self-administered drug exposure (Lobo et al., 2013). To determine the behavioral consequences of cell type–specific induction of ΔFosB in the NAc, viral-mediated gene transfer was used to overexpress ΔFosB in D1 or D2 MSNs (Grueter et al., 2013). It was found that overexpression in D1 MSNs enhanced cocaine sensitization and CPP, whereas overexpression in D2 MSNs had no measured behavioral consequences (Grueter et al., 2013).

In addition to BAC transgenic mice, D1- and D2-Cre mice have been used to selectively express a number of exogenous proteins specifically in either MSN population and to create cell type–specific knockout animals. Using this strategy to selectively delete the brain-derived neurotrophic factor TrkB receptor in D1 or D2 MSNs, Lobo et al. (2010) demonstrated opposing effects on cocaine reward when measured by an unbiased CPP procedure, with the loss in D1 cells promoting and the loss in D2 cells reducing preference scores. Direct optogenetic activation of D1 or D2 MSNs similarly modulated cocaine reward in opposing directions (Lobo et al., 2010).

Overall, the emerging literature using these D1 and D2 transgenic mice supports a role for D1 MSNs in positively regulating psychostimulant-induced behavioral and cellular responses and D2 MSNs in negatively regulating these behaviors (Bertran-Gonzalez et al., 2008; Hikida et al., 2010; Lobo et al., 2010; Ferguson et al., 2011; Bock et al., 2013; Farrell et al., 2013; Park et al., 2013). Although the literature has been consistent in this regard, an important caveat that is discussed in more detail in this section is that although behaviors coded by D1 and D2 MSNs are traditionally interpreted as mediated by the direct and indirect pathways, respectively, D1 and D2 accumbens MSNs send a mixed projection to the VP, making the classic interpretation of direct and indirect pathways at least partly incorrect (Kupchik et al., 2015).

B. Interneurons

The 5%–10% of cells in the accumbens that are not MSNs are broadly classified as interneurons, and they can be chemically coded into several classes by their protein expression profile (Fig. 2) (Kawaguchi et al., 1995). Three discrete types of GABAergic interneurons are in the striatum: those that express parvalbumin; those that coexpress somatastatin, neuropeptide Y, and neuronal nitric oxide synthase (nNOS); and those that express calretinin (Tepper et al., 2010). Although parvalbumin- and calretinin-containing interneurons have been anatomically identified, their role in the physiology of drug addiction remains to be clearly elucidated and they are not discussed further. The fourth class of interneurons is cholinergic and is characterized by expression of choline acetyltransferase and relatively large soma.

C. Glial Cells

Astrocytes regulate glutamatergic synaptic plasticity within the accumbens by controlling the extracellular glutamate concentration via coordinated uptake and release (Kalivas, 2009). Glial cells release glutamate in a variety of ways (Scofield and Kalivas, 2014), including through the cystine-glutamate exchanger (system x_c-) (Malarkey and Parpura, 2008). System x_c- catalyzes the 1:1 release of astrocytic glutamate in exchange for extracellular cystine, a mechanism that provides more than 50% of the extrasynaptic glutamate measured in the NAcore via in vivo microdialysis (Hascup et al., 2008; van der Zeyden et al., 2008). Interestingly, chronic cocaine and nicotine exposure downregulate expression of x_c- (Moran et al., 2003; Kalivas, 2009; Knackstedt et al., 2009), which serves as a plausible mechanistic explanation for the decrease in basal glutamate levels observed after chronic exposure to these drugs (Table 1). The maintenance of extracellular glutamate levels through system x_c- is of central importance in the regulation of synaptic plasticity in the corticoaccumbens circuit because it provides tone on presynaptic mGluR2/3 autoreceptors that regulate the synaptic release of glutamate (Moran et al., 2005). As such, drug-induced reduction of glutamate tone onto mGluR2/3 promotes synaptic potentiation and enhances glutamate release induced by conditioned cues and drug exposure. This potentiated release causes synaptic glutamate spillover and access of extracellular glutamate to postsynaptic glutamate receptors, which engages synaptic plasticity responsible for drug-seeking behavior (Kalivas, 2009).

After neuronal synaptic glutamate release, astrocytes terminate signaling by removing glutamate from the synaptic cleft through the patterned expression of the Na⁺-dependent glial glutamate transporter (GLT)-1 (EAAT2) (Williams et al., 2005). This glial function is required for the fidelity of glutamatergic synaptic communication and protection from excitotoxicity, since GLT-1 is responsible for more than 90% of uptake in the brain (Danbolt, 2001). Chronic exposure to several classes of addictive substances, including cocaine, nicotine, ethanol, and heroin, reduces expression of GLT-1 (Knackstedt et al., 2010a; Sari and Sreemantula, 2012; Gipson et al., 2013b; Shen et al., 2014b; Reissner et al., 2015). As such, GLT-1 downregulation may serve as a common drug-induced neuroadaptation contributing to relapse vulnerability. Mechanistically, lack of glutamate uptake in the NAc promotes spillover of synaptically released glutamate out of the synaptic cleft and into the extracellular space, causing the activation of postsynaptic glutamate receptors responsible for the rapid transient synaptic potentiation associated with relapse (see the detailed discussion in section V).

D. Extracellular Matrix

A proteinacous network of secreted macromolecules deemed the extracellular matrix (ECM) supports the complex architecture of interconnected neural and glial processes in the neuropil. The ECM comprises approximately 20% of the volume of the mature brain (Nicholson and Syková, 1998) and has a highly organized composition consisting of two main classes of proteins: glycosaminoglycans normally linked to proteins in the form of proteoglycans and fibrous proteins including laminin, collagen, elastin, and fibronectin. The ECM not only serves as a structural anchor for neurons and glia, but it is also a signaling domain that regulates neurotransmission, cellular growth, plasticity, and apoptosis/survival signaling (Lee et al., 2001; Gu et al., 2002; Verslegers et al., 2013). Furthermore, signaling between neurons and the ECM is bidirectionally transduced; that is, changes in the extracellular milieu can affect intracellular signaling (outside-in signaling), and changes in the intracellular environment can be transduced to signaling within the ECM (inside-out signaling).

All parts of the tripartite synapse (presynapse, postsynapse, and glia) interact either directly or indirectly with the ECM (Dityatev and Rusakov, 2011). One such method for interaction is through cell adhesion molecules (CAMs), which are transmembrane proteins that bind to ECM glycoproteins and also to intracellular signaling molecules to organize the synaptic interface and regulate synaptic activity (Shinoe and Goda, 2015). The integrins and the intracellular CAMs are the most well studied CAMs in the brain (Wiggins et al., 2011; Niedringhaus et al., 2012; Lonskaya et al., 2013). Because the ECM can respond to activity in the other three compartments of the tripartite synapse, it is now considered a fourth synaptic compartment, causing the emergence of the term tetrapartite synapse (for review, see Smith et al., 2015a). For example, activity-dependent ECM signaling can liberate latent growth factors (Saygili et al., 2011), affect synaptic and extrasynaptic receptor content (Michaluk et al., 2009), and stimulate morphologic and physiologic synaptic plasticity (Wang et al., 2008).

The ECM must be degraded to allow morphologic plasticity of dendritic spines (discussed in greater detail in section V), making catabolic enzymes essential for the induction of classic forms of synaptic plasticity, such as LTP (Wang et al., 2008; Szepesi et al., 2013). MMPs are the family of zinc-dependent endopeptidases that degrade ECM proteins and are permissive to a number of events classically associated with synaptic plasticity, including morphologic changes in dendritic spines (Michaluk et al., 2011; Stawarski et al., 2014; Verslegers et al., 2015), NMDAR lateral diffusion, and AMPAR phosphorylation and insertion (Michaluk et al., 2009; Szepesi et al., 2014). Specifically, MMP-2 and MMP-9 play important roles in aberrant synaptic plasticity associated with neurologic disorders (Mizoguchi et al., 2011; Stawarski et al., 2014), with recent research demonstrating the importance of MMPs in drug behavioral effects and relapse. For example, inhibiting MMP-9 attenuates cocaine CPP (Brown et al., 2008), and MMP-9 is involved in regulating synaptic plasticity underlying acquisition of nicotine CPP (Natarajan et al., 2013). In addition, MMP-9 activation is implicated in the development of morphine tolerance (Nakamoto et al., 2012). MMP activity in the hippocampus (namely, MMP-9 activity) is disrupted after ethanol exposure and thereby impairs acquisition of a spatial memory task (Wright et al., 2003). Interestingly, MMP activity is associated with the transient plasticity found during cue-induced cocaine reinstatement (Smith et al., 2014). Specifically, MMP-2 activity in the NAcore is constitutively elevated after extinction of cocaine self-administration, and inhibiting this activity reversed cocaine-induced potentiation of the spine head diameter and the AMPA/NMDA ratio. Furthermore, MMP-9 activity is transiently increased during cue-induced cocaine reinstatement, and blockade of this induction also blocks the transient synaptic potentiation, which accompanies reinstatement (Smith et al., 2014). In addition, cue-induced heroin and nicotine seeking increases MMP-2/MMP-9 activity, and MMP activity is also required for synaptic plasticity underlying escalation of ethanol intake after chronic exposure (Smith et al., 2011).

MMP-2 and MMP-9 are unique within the metalloproteinase superfamily for their ability to recognize and expose arginine-glycine-aspartate domains that are endogenous ligands at the integrin family of CAMs (Verslegers et al., 2013). Application of recombinant, autoactive MMP-9 to hippocampal slices drives LTP of field potentials and spine head enlargement even in the absence of high-frequency stimulation, and this effect is occluded by a β1-integrin blocking antibody (Wang et al., 2008). Integrins are coupled to the integrin-linked kinase, which can phosphorylate GluA1 at Serine 845, driving the Ca²⁺-permeable (CP) AMPARs into the synapse (Chen et al., 2010). Integrin-linked kinase can also phosphorylate cofilin to stimulate actin polymerization and dendritic spine enlargement (Kim et al., 2008). The β3-integrin subunit is increased in the PSD subfractionation of rats that have undergone 21 days of extinction of cocaine self-administration, whereas expression of the β1-subunit remains unchanged (Wiggins et al., 2011). The β3 subunit is physically coupled to AMPARs via their cytoplasmic domains (Pozo et al., 2012) and is required for activity-dependent synaptic scaling of glutamatergic synapses (Cingolani et al., 2008). For more information on the ECM, MMPs, and tissue inhibitors of MMPs, see Seals and Courtneidge (2003), Brew and Nagase (2010), Huntley (2012), Oohashi et al. (2015), Singh et al. (2015), Smith et al. (2015a,b), and Vafadari et al., (2015).

A. Nucleus Accumbens Core

The NAcore is responsible for the evaluation of reward and initializing reward-related motor action (Voorn et al., 2004; Sesack and Grace, 2010; Shiflett and Balleine, 2011). It serves as an intermediate between the NAshell responsible for reward prediction and reward learning and the dorsolateral striatal regions responsible for the encoding and execution of learned habits, skills, and action sequences (Shiflett and Balleine, 2011). The NAcore is essential for acquiring drug-taking behaviors and cue-elicited drug-seeking responses. For psychostimulant drugs, learning drug reward associations is largely dependent on dopaminergic and glutamatergic signaling within the NAcore, whereas reinstatement is mostly driven by glutamate (Kalivas and Volkow, 2005; Koob and Volkow, 2010). However, it is important to note that additional neurochemical mechanisms are involved in drug reward associations and reinstatement of nonpsychostimulant drugs such as opiates and benzodiazepines (for review, see Badiani et al., 2011; Nutt et al., 2015).

B. Nucleus Accumbens Shell

The NAshell is the primary striatal region involved with motivation and reward-related processes. Akin to nonstriatal basal ganglia nuclei, the shell is heavily interconnected with regions such as the lateral hypothalamus and extended amygdala and is therefore often considered a transition zone that serves as a point of convergence between these systems (Sesack and Grace, 2010). It is thus ideally positioned to process motivationally relevant information in accordance with autonomic, emotional, and basal ganglia systems (Heimer et al., 1997).

A. Long-Term Synaptic Plasticity

One of the more robust features of addiction is the enduring propensity to relapse. This persistent state was long hypothesized to be encoded by synaptic changes in the mesolimbic system. Indeed, early work in the VTA shows synaptic adaptations occurring after exposure to cocaine or morphine (Bonci and Williams, 1996, 1997; Ungless et al., 2001; Thomas et al., 2008). However, the desire to use drugs is encoded in glutamatergic synapses of the NAc (Kalivas, 2009). The best established data set for drug-induced synaptic plasticity in the NAc is after cocaine use. A single noncontingent injection of cocaine does not produce any synaptic changes in excitatory transmission in the NAc, whereas repeated injections cause a depression of EPSCs (Thomas et al., 2001; Kourrich et al., 2007; Huang et al., 2009; Ortinski et al., 2012). A similar depression is also seen after self-administrated cocaine (Schramm-Sapyta et al., 2006). Interestingly, a period of withdrawal from cocaine leads to potentiation of excitatory input, be it after a single cocaine injection (Pascoli et al., 2012), repeated cocaine injections (Kourrich et al., 2007; Britt et al., 2012), cocaine self-administration (Gipson et al., 2013a; Pascoli et al., 2014), or during the incubation of craving (Conrad et al., 2008).

Evidence for other addictive drugs is not complete and at times shows opposite changes in the NAc compared with cocaine. For example, withdrawal from nicotine self-administration shows a similar potentiation (Gipson et al., 2013b), whereas the results are mixed for studies examining the NAc after withdrawal from heroin exposure (Russo et al., 2010; Shen et al., 2011; Wu et al., 2012). Chronic ethanol induces an increase in mEPSC frequency with no change in amplitude; however, mEPSC frequency is decreased and mEPSC amplitude is increased after withdrawal, indicating that two opposing mechanisms are activated (Spiga et al., 2014). Regardless of the specific change and the specific model used, these data support the perspective that the enduring symptoms of drug addiction may be encoded by synaptic changes in the NAc.

B. Short-Term Synaptic Plasticity

The long-term changes described above are all induced by past exposure to drugs. Thus, they may make the addict susceptible for relapse. Importantly, since the enduring synaptic plasticity outlined above is not induced by all addictive drugs, it may not reflect the key adaptations that underpin the engagement of drug-seeking behaviors that mediate relapse. Thus, it is possible that when an animal engages in drug-seeking behaviors, additional synaptic plasticity may occur that mediates the behavior. These changes would need to be rapidly induced, given that drug-seeking behavior can be rapidly initiated by drug-associated cues and, if relevant to the behavior, should be shared across chemical classes of addictive drug.

Substantial evidence supports the likelihood that glutamate neurotransmission in the NAc is critical for drug seeking. For example, both pharmacological (Cornish and Kalivas, 2000; Di Ciano and Everitt, 2001; Park et al., 2002; Kalivas et al., 2005) and optogenetic (Stefanik and Kalivas, 2013; Stefanik et al., 2013b) inhibition of corticoaccumbens projections attenuate the reinstatement of drug-seeking behavior. To determine whether this necessary glutamate transmission is associated with alterations in synaptic strength, we recently examined excitatory synaptic transmission in the NAcore at different times after a rat was exposed to a drug-associated cue that reinstates cocaine-seeking behavior (Gipson et al., 2013a). We found that the glutamatergic input to the NAc from the PLC, which is already potentiated during withdrawal, is further potentiated already by 15 minutes after cue-induced reinstatement. Notably, the amount of lever presses during the reinstatement session was positively correlated with the increase in AMPA/NMDA (Gipson et al., 2013a) and this correlation is the strongest when AMPA/NMDA is correlated with the behavior during the first 5 minutes of the reinstatement session (Gipson et al., 2014). Also, akin to the behavioral response, the AMPA/NMDA ratio is back to baseline levels by the end of the 120-minute reinstatement session. Importantly, transient synaptic potentiation is also found after reinstatement of nicotine (Gipson et al., 2013b) and heroin (Shen et al., 2011) seeking. Thus, the cue-induced synaptic potentiation observed during reinstatement may be a common phenomenon across classes of addictive drugs, and thereby has the potential to provide targets for treating relapse to drug use.

C. Morphologic Plasticity

Drugs of abuse have been found to alter dendritic spine morphology on MSNs within both the NAcore and NAshell. Dendritic spines are very plastic (Nimchinsky et al., 2002), and changes in their structure are generally accepted to be strongly associated with synaptic strength since their spontaneous generation, selection, and consolidation underlie the physical foundation for learning and memory (De Roo et al., 2008; Kasai et al., 2010a,b; Dietz et al., 2012). In general, the formation of new spines or enlargement of existing spines is considered a correlate of LTP, whereas the retraction or contraction of spines is associated with LTD (Fig. 3). Measurement of dendritic spine morphologic characteristics, such as density, volume, head diameter, and neck length, involves using multiple methods that allow for either two- or three-dimensional analysis of spines on dendritic branches, including filling cells with lucifer yellow, the lipophilic dye DiI, and Golgi-Cox staining, among others (Russo et al., 2010). More recently, two-photon imaging allows for real-time visualization of spine dynamics in vivo using a cranial window (although this technique is limited to superficial layers of the neocortex) (Isshiki and Okabe, 2014; Isshiki et al., 2014). Although each method has benefits and drawbacks, visualizing dendritic spine morphology has advanced our understanding of drug-induced alterations in postsynaptic spines within addiction circuitry.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 3.

Spine head diameter and synaptic potentiation. Synaptic plasticity involves both structural and functional changes that allow stronger or weaker synaptic connections. In LTP, spine head diameter increases to allow insertion of AMPARs at the synapse. The functional output of synaptic potentiation is an increase in the ratio between AMPA and NMDA EPSCs, with either more AMPA or less NMDA. For changes in spine morphology to occur, the actin cytoskeleton must grow and become more complex to allow structural growth or shrinkage. Actin cycling involves the formation of filamentous actin from the monomer (G-actin). These filaments have barbed ends and are organized into long stalks that cycle to expand or contract dendritic spines. In LTD, spine head diameter decreases and AMPARs are removed. In parallel with these structural changes, the functional reading of synaptic plasticity, the AMPA/NMDA ratio, is decreased.

Interestingly, complex changes in excitatory neurotransmission have been found in the NAcore (Grueter et al., 2012). In addition, different drugs of abuse (e.g., heroin and cocaine) alter dendritic spine morphology differentially, such that after extended withdrawal (2 to 3 weeks) from heroin or morphine, dendritic spines quantified via density or head diameter rest in a depressed state (Robinson and Kolb, 1999; Shen et al., 2011); after withdrawal from cocaine or nicotine, spines rest in a relatively potentiated state, measured as increased density, head diameter, or neck length (Brown and Kolb, 2001; Robinson et al., 2001; Gipson et al., 2013a,b) within the NAcore or NAshell. Thus, the enduring change (increase or decrease in head diameter) in synaptic strength inferred from the morphology of dendritic spines is not consistent across different drug classes. However, in the case of heroin, nicotine, and cocaine, reinstatement of drug seeking elicits similar increases in head diameter after contingent exposure to discrete cues or environmental context associated with the drug of abuse [cocaine (Gipson et al., 2013a; Stankeviciute et al., 2014) or nicotine (Gipson et al., 2013b)] or priming of the drug itself [heroin (Shen et al., 2011) or cocaine (Shen et al., 2009, 2014a]. Thus, similar to the electrophysiological plasticity estimated by AMPA/NMDA ratios, relapse-associated increases in spine head diameter are a consistent neuroadaptation and may mediate the shared characteristic of relapse vulnerability between drug classes. In contrast, constitutive changes vary between drug classes and are less likely to underpin the shared behavioral characteristics of addiction, such as drug relapse.

Although both NAcore and NAshell MSNs show similar general changes to treatment with cocaine, detailed evaluation suggests that cocaine differentially regulates synaptic plasticity between these two subregions in distal versus proximal dendrites (Dumitriu et al., 2012). For example, at 4 hours of withdrawal from cocaine injection, proximal spine density is increased in the shell but not core. Furthermore, at 24 hours of withdrawal, an increase in proximal dendritic spine density is again found in the shell but not core. After 28 days of withdrawal, spine density in the core remained decreased but returned to baseline in the shell. In contrast with the these more subtle differences in constitutive cocaine-induced changes in spine morphology, the accumbens subcompartments diverge markedly in the induction of transient potentiation where the NAcore shows potentiation but the NAshell does not respond to a cocaine cue (Smith et al., 2014).

D. Functional Relevance of Spine Dynamics

The mechanisms by which spines grow or shrink have been extensively studied, and bigger dendritic spines have been associated with stronger dendritic contacts (Kopec and Malinow, 2006). Actin is a main structural component of dendritic spines and is organized into filaments that are associated with the plasma membrane and at the synapse. These filaments have barbed ends and are organized into long stalks that cycle to expand or contract dendritic spines (Fifková and Delay, 1982; Matus et al., 1982, 2000; Fifková and Morales, 1992). Activation of AMPARs increases head diameter (Zhao et al., 2012), and this is attributed to a stabilization of spines through actin-dependent mechanisms (Fischer et al., 2000; Richards et al., 2004). Specifically, this is thought to be due to a shift in the balance between the two forms of actin: F-actin (filament) and G-actin (monomer) (Zhao et al., 2012). Indeed, remodeling of actin underlies morphologic changes in spines during synaptic plasticity, and this process constantly reshapes adult brain circuitry and connections in response to environmental stimuli; in turn, this underlies learning and memory processes. Activation of AMPARs during the induction of LTP has been shown to increase head diameter (Fischer et al., 2000), and an increase in the GluA1 subunit of the AMPAR is positively correlated with the ability of a calcium transient produced in the head of the spine to diffuse into the dendrite (Korkotian and Segal, 2007). In addition, short spines had a higher probability of raising GluA1 than long ones, indicating functional relevance for morphologic differences in spine shape, including length. The implication that morphologic changes in spines drive changes in synaptic AMPAR expression is a supported by pharmacological inhibition of F-actin altering the movement of receptors into and out of the synapse (Charrier et al., 2006; Cingolani and Goda, 2008). In addition, manipulation of F-actin via overexpression of Drebrin-A, an abundant neuron-specific F-actin binding protein, augmented glutamatergic transmission measured as a change in the amplitude and frequency of spontaneous AMPA currents in mature cultured hippocampal neurons (Ivanov et al., 2009a,b). As well, an increase in head diameter has been hypothesized to be the result of increased actin cycling and AMPAR trafficking to the cell surface (Kopec and Malinow, 2006; Kopec et al., 2006). Activation of F-actin via tetanic stimulation caused a rapid, persistent shift toward F-actin from G-actin and increased CaMKII levels. CaMKII is essential for recruiting AMPARs into the postsynaptic membrane (Okamoto et al., 2004) and is necessary for induction of NAshell dendritic spines and behavioral sensitization to cocaine (Robison et al., 2013). Taken together, these results imply that activation of F-actin could increase AMPARs in the postsynaptic membrane, and spine enlargement may be required to allow AMPAR insertion in cocaine-induced synaptic plasticity.

Chronic cocaine or morphine exposure is associated with an increase in F-actin and actin cycling (Toda et al., 2006). Manipulation of the mechanisms of spine enlargement during withdrawal from cocaine exposure has shown that compared with saline (drug-naïve animals), animals withdrawn from chronic cocaine had elevated levels of F-actin in the NAc (both core and shell) (Shen et al., 2009). Furthermore, animals given a cocaine injection after withdrawal from chronic experimenter-delivered cocaine showed a transient but robust increase in F-actin and Arp-3 (PSD protein regulating actin cytoskeleton cycling). In addition, latrunculin A, which binds to G-actin and prevents polymerization of G-actin into F-actin, has been shown to inhibit F-actin levels proportionally to the rate of F-actin disassembly (Morton et al., 2000; Toda et al., 2006). When latrunculin was microinjected into the NAcore, it reduced spine density and caused a corresponding decrease in F-actin and PSD-95 in the postsynaptic density of cocaine-withdrawn but not drug-naïve animals. Latrunculin also abolished the increase in NAcore head diameter and behavioral sensitization (as measured via locomotor activity). Surprisingly, latrunculin microinjection into the NAcore potentiated cocaine-induced reinstatement, indicating that the increase in F-actin after cocaine withdrawal may be compensatory relative to drug-seeking behavior (Toda et al., 2006, 2010). In a similar line of research, others found that inhibition of actin cycling in the amygdala selectively disrupted methamphetamine-associated memory in methamphetamine CPP and contextual renewal of methamphetamine seeking (Young et al., 2014).

Recent technologies allow us to determine cell-type specificity of spine morphology, most often using BAC transgenic mice that selectively label D1- or D2-expressing MSNs and viral vectors that selectively target these cell subpopulations. A majority of studies show that cocaine-induced structural plasticity and synaptic plasticity alterations in the NAc are preferentially observed in or are more persistent in D1 MSNs (Golden and Russo, 2012). With prolonged, repeated noncontingent cocaine treatment, there is a selective increase in dendritic spine density in D1 MSNs in the NAc (core and shell) with an increase in spine diameter in the NAcore during early but not late withdrawal (Dobi et al., 2011). These results were indirectly corroborated by a study showing that D1 receptor knockout mice fail to display cocaine-induced morphologic changes; D1 receptor but not D2 receptor antagonists likewise prevented the increase in spine density, although the cell-type specificity of these changes was not investigated (Ren et al., 2010). In contrast, others have reported that repeated cocaine treatment increases dendritic spine density in both cell types (Lee et al., 2006; Li et al., 2012), although these changes still only persist in D1 MSNs (Lee et al., 2006). Inconsistencies between the various reports of noncontingent cocaine delivery may be attributed to a variety of factors, including drug dose, withdrawal time, and analysis method. A number of reports indicate that cocaine-induced behaviors, including seeking and sensitization, are mediated by activation of D1 MSNs (Ferguson et al., 2011; Lobo and Nestler, 2011; Bock et al., 2013; Smith et al., 2013). Pertinent to mechanism, cotransducing the NAshell of the BAC transgenic mice with the Cre-dependent herpes simplex virus (HSV)–mCherry and HSV–green fluorescent protein–ΔFosB allowed for analysis of spine morphology alterations by ΔFosB. Acute drug exposure (including most drugs of abuse) has been shown to induce the long-lasting accumulation of ΔFosB in the NAc (Nestler, 2008), and noncontingent cocaine-induced alterations in spine morphology have been shown to be dependent on ΔFosB (Maze et al., 2010). Using transgenic mice, ΔFosB was found to selectively increase dendritic spine density in D1- but not D2-expressing MSNs after repeated injections of noncontingent cocaine (Grueter et al., 2013). The cell-type specificity of the other molecular mechanisms underlying drug-induced plasticity summarized above is yet to be investigated.

A. α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Receptors

As discussed above, in the accumbens, activation of AMPARs is required for the acute excitation of MSNs by glutamatergic inputs that are required to induce drug seeking (Wolf and Ferrario, 2010). Systemic delivery of AMPAR antagonists inhibits cue-induced cocaine (Bäckström and Hyytiä, 2003) and ethanol (Bäckström and Hyytiä, 2004) seeking, as well as methamphetamine (Miyatake et al., 2005) and amphetamine (Mead and Stephens, 1999) CPP and the induction and expression of amphetamine behavioral sensitization (Karler et al., 1991). Evidence suggests that the efficacy of the systemic delivery of AMPA antagonists is enacted at least in part by glutamatergic neurotransmission in the accumbens, because systemic administration of the AMPAR antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dion (NBQX) inhibited cue-induced reinstatement of cocaine seeking and was accompanied by decreased activity in NAcore neurons (Zavala et al., 2008).

Infusion into the NAc of the glutamate analog AMPA (which serves as a selective AMPAR agonist) alone initiates cocaine seeking to levels that parallel reinstated drug seeking precipitated by a noncontingent injection of cocaine (Ping et al., 2008). In these experiments, AMPA infusion into the NAshell was more effective at producing cocaine-seeking behavior than infusions made into the NAcore, yet both produced a significant effect (Ping et al., 2008). The importance of AMPARs in cocaine seeking is further illustrated by downregulating the AMPAR subunit GluR1 mRNA in the accumbens using an oligonucleotide antisense strategy to decrease both cocaine- and AMPA-primed reinstatement of cocaine seeking. This effect is also observed when inhibitory nucleic acid is delivered to either the NAcore or NAshell (Ping et al., 2008).

Cocaine seeking can be induced through the microinfusion of cocaine into the mPFC, and this behavior is blocked by infusion of the AMPAR antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) into the accumbens (no distinction between NAcore and NAshell) (Park et al., 2002). When CNQX is delivered directly to the NAcore, the motor stimulant effect of a cocaine injection in cocaine-sensitized animals is inhibited (Pierce et al., 1996), as is responding during cocaine self-administration (Cornish et al., 1999; Suto et al., 2009) and intake during extended access to cocaine (Doyle et al., 2014). Furthermore, infusion of CNQX into the NAcore reduces cue-induced (Bäckström and Hyytiä, 2007), context-induced (Xie et al., 2012), and cocaine-primed (Cornish and Kalivas, 2000; Famous et al., 2008) reinstatement of cocaine seeking. AMPAR blockade–mediated inhibition of cocaine seeking disrupts the efficacy of a conditioned cue to engage cocaine seeking, because delivery of the AMPAR antagonist LY293558 [(3S,4aR,6R,8aR)-6-[2-(1H-tetrazol-5-yl)ethyl]decahydroisoquinoline-3-carboxylic acid] into the NAcore decreases active lever responding, yet increases inactive lever responding (Di Ciano and Everitt, 2001). Infusion of CNQX into the NAcore also inhibits both cue-induced and drug-primed heroin seeking (LaLumiere and Kalivas, 2008). However, infusion of CNQX into the accumbens (no distinction made between the NAcore and NAshell) attenuates the locomotor response to a d-amphetamine administration in animals conditioned by previous d-amphetamine exposure (Burns et al., 1994).

As discussed above, accumbens MSNs express increased levels of CP-AMPARs after the incubation of cocaine craving. Interestingly, NAc infusion of Naspm, a selective antagonist of GluR2-lacking CP-AMPARs, inhibits cued cocaine seeking, demonstrating the importance of this drug-induced alteration of the AMPAR subunit expression profile (Conrad et al., 2008). Moreover, cocaine-induced reinstatement of lever pressing is associated with a transient increase in AMPARs and this is prevented by pretreatment into the NAcore or NAshell with a cell-permeable peptide (Pep2-EVKI) that disrupts GluR2 trafficking to the membrane (Famous et al., 2008).

B. N-Methyl-d-Aspartate Receptors

As discussed above, NMDARs serve as key regulators of the synaptic plasticity linked to the neurologic processes controlling learning and memory (Morris, 2013), with pharmacological blockade of NMDARs being a common mechanism of action for dissociative anesthetic drugs including ketamine and dizoclipine (Mion and Villevieille, 2013). Systemic administration of the noncompetitive NMDAR antagonist dizoclipine (MK-801 or [5R,10S]-[+]-5-methyl-10,11- dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine) disrupts the reconsolidation of cocaine context-associated memory prior to testing for a CPP (Brown et al., 2008), yet it has no effect on cocaine-primed reinstatement. However, when cocaine is not on board, systemic administration of MK-801 dose-dependently reinstates cocaine seeking in extinguished animals (De Vries et al., 1998). Systemic delivery of MK-801 also inhibits nicotine-induced sensitization of locomotor activity (Shoaib and Stolerman, 1992) and amphetamine CPP (Table 2) (Tzschentke, 2007). However, MK-801 is possibly reinforcing, because studies show that MK-801 produces a CPP when given alone to naïve mice (Panos et al., 1999). Furthermore, animals will directly self-administer MK-801 microinfusions into the NAshell, but not the NAcore (Carlezon and Wise, 1996).

Another pharmacological agent that inhibits NMDAR signaling (although it has also been shown to activate GABA_A receptor signaling; Williams, 2005) is N-acetyl homotaurine (acamprosate), which is used to treat alcohol withdrawal in humans (Franck and Jayaram-Lindström, 2013). In preclinical studies, systemic administration of acamprosate inhibits cue-induced and drug-primed cocaine seeking (Bowers et al., 2007), cue-induced nicotine seeking (Pechnick et al., 2011), as well as cocaine and ethanol CPP (McGeehan and Olive, 2003a) and the reinstatement of cocaine CPP (McGeehan and Olive, 2006). Interestingly, acamprosate inhibits morphine-induced sensitization (but does not inhibit stress or drug-primed reinstatement of heroin seeking), an effect that is accompanied by reduced dopamine levels in the NAshell (Spanagel et al., 1998). Similar results are obtained in ethanol studies in which acamprosate inhibits ethanol intake and CPP, which is also associated with reduced levels of dopamine release in the NAshell (Olive et al., 2002). Studies performed in neocortical cultures suggest that acamprosate treatment exerts its therapeutic effect, at least in part, through preventing glutamate excitotoxicity during ethanol withdrawal (al Qatari et al., 2001)

Yet another pharmacological agent that inhibits activation of NMDARs is memantine, which is commonly used in the treatment of Alzheimer’s disease as a means of inhibiting neuronal excitotoxicity (Zádori et al., 2014). When given systemically, memantine inhibits morphine (Ribeiro Do Couto et al., 2004) and cocaine (Kotlińska and Biała, 2000) CPP, as well as nicotine but not cocaine self-administration (Blokhina et al., 2005). Studies show that memantine treatment also reverses cocaine-induced reductions in the expression of tumor necrosis factor-α in the NAc of animals that show inhibited cocaine CPP (no distinction made between the NAcore and NAshell) (Lin et al., 2011).

Accumbens NMDAR-dependent plasticity is required for the early stages of learning. Accordingly, studies show that blockade of accumbens NMDARs inhibits the acquisition of an operant sucrose self-administration task, yet it has no effect on lever pressing for sucrose once the task is learned (Kelley et al., 1997). Studies show that infusion of (2R)-amino-5-phosphonovaleric acid (AP5) into the NAcore dose-dependently inhibits cocaine-induced locomotion, whereas infusion of AP5 into the NAshell has no effect. However, the same group also reports that infusion of AP5 into the NAshell produces an increase in spontaneous locomotion, whereas infusion into the NAcore has no effect on activity (Pulvirenti et al., 1994). Infusion of AP5 in the NAcore or NAshell also enhances context-induced, cocaine-conditioned locomotion (Rodríguez-Borrero et al., 2006). Interestingly, contradictory evidence for the role of AP5 infusion on reinstated cocaine seeking exists, with one report demonstrating that AP5 infusion into the NAcore or NAshell induces reinstated cocaine seeking, with the NAshell infusion having the stronger effect (Famous et al., 2007), and the other group demonstrating that NMDAR blockade via AP5 infusion into the NAcore dose-dependently inhibits cue-induced cocaine seeking (Bäckström and Hyytiä, 2007). One important consideration is that Famous et al. (2007) used a higher dose of AP5 (3 and 30 μg) to promote reinstated cocaine seeking, whereas Bäckström and Hyytiä (2007) found that lower doses of AP5 (1 and 2 μg) inhibit cue-induced cocaine seeking. Infusion of AP5 into the accumbens (no distinction made between the NAcore and NAshell) also decreases the potentiation of conditioned reinforcement caused by d-amphetamine (Burns et al., 1993) and decreases oral ethanol self-administration (Rassnick et al., 1992).

Systemic administration of the GluN2B-containing NMDAR subtype–specific antagonist ifenprodil inhibits cue- and drug-induced heroin seeking (Shen et al., 2011), cue-induced nicotine seeking (Gipson et al., 2013b), as well as morphine (Suzuki et al., 1999; Ma et al., 2011) and methamphetamine (Miyatake et al., 2005) CPP. Direct infusion of ifenprodil or GluN2B-specific small interfering RNA (siRNA) into the NAcore inhibits cue-induced and drug-primed heroin seeking (Shen et al., 2011). Similarly, infusion of GluN2B-specific siRNA into the NAshell inhibits morphine CPP (Kao et al., 2011), whereas infusion of GluN2B-specific siRNA into the NAcore inhibits cue- and drug-induced heroin seeking (Shen et al., 2011). These data illustrate the importance of the GluN2B-containing NMDAR subtype in mediating accumbens glutamatergic plasticity that underlies opiate reward and drug seeking.

C. Group I Metabotropic Glutamate Receptors (Metabotropic Glutamate Receptors 1 and 5)

Studies show that systemic blockade of postsynaptic Gq-coupled mGluR1 receptors with the antagonist JNJ-16259685 (3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone) inhibits cocaine behavioral sensitization (Dravolina et al., 2006) as well as cocaine and methamphetamine intake (Achat-Mendes et al., 2012). Furthermore, direct infusion of the same drug (JNJ-16259685) into the NAcore inhibits context-induced cocaine seeking in the drinking-in-the-dark paradigm (Xie et al., 2012), whereas blockade of mGluR1 in the NAshell inhibits ethanol intake in mice.

As described above, extended access cocaine self-administration followed by incubation of craving (approximately 30 days of forced abstinence, without extinction training) dramatically increases drug-seeking behavior and markedly decreases surface expression mGluR1 receptors in the NAc. This incubation-mediated regulation of mGluR1 leads to the accumulation of CP- AMPARs. Using this model, restoration of mGluR1 tone with positive allosteric modulators such as Ro67-7476 [(2S)-2-(4-fluorophenyl)-1-[(4-methylphenyl)sulfonyl]-pyrrolidine] or SYN119 [9H-Xanthene-9-carboxylic acid (4-trifluoromethyl-oxazol-2-yl)-amide], given either systemically or directly in the NAcore, inhibits cued cocaine seeking (Loweth et al., 2014). Importantly, SYN119 treatment also restored the altered rectification index in electophysiological recordings, indicating that the restoration of mGluR1 tone reverses the accumulation of CP-AMPARs in the accumbens (Loweth et al., 2014).

Like mGluR1, mGluR5 receptors are also Gq coupled and are preferentially localized postsynaptically (Shigemoto et al., 1997). Systemic administration of mGluR5 antagonists inhibits cocaine self-administration (Tessari et al., 2004), CPP (McGeehan and Olive, 2003b), and cue- and drug-induced reinstatement of cocaine seeking (Kumaresan et al., 2009), as well as nicotine self-administration and drug-primed reinstatement (Tessari et al., 2004). Systemic administration of mGluR5 antagonists also inhibits morphine (Popik and Wróbel, 2002) and amphetamine (Herzig et al., 2005) CPP. In addition, systemic administration of fenobam (a mGluR5 negative allosteric modulator) inhibits cue- and drug-induced methamphetamine seeking (Watterson et al., 2013), cocaine intake, and cue- and drug-induced cocaine seeking (Keck et al., 2013). However, like the mGluR2/3 agonist discussed below, both groups found fenobam to reduce sucrose seeking (Keck et al., 2013; Watterson et al., 2013). Additional negative allosteric modulators of mGluR5 are currently under development, including MFZ 10-7 (3-fluoro-5-[2-(6-methyl-2-pyridinyl)ethynyl]benzonitrile hydrochloride), which inhibits cocaine intake as well as cue-induced and drug-primed reinstatement (Keck et al., 2014). Keck et al. report that although 3-((2-methyl-4-thiazolyl)ethynyl)pyridine (MTEP) and MFZ 10-7 lowered rates of sucrose intake, they did not affect overall sucrose intake or locomotor activity.

Studies in animal models of addiction indicate that in the NAcore, the effect of activating postsynaptic Gq-coupled mGluR5 receptors is opposite of that of activating presynaptic mGluR2/3 receptors (Kalivas, 2009; Moussawi and Kalivas, 2010). Infusion of (S)-3,5-dihydroxyphenylglycine (group I mGluRs) or 2-chloro-5-hydroxyphenylglycine (specific mGluR5 agonist) into the NAcore promotes the reinstatement of cocaine seeking (Wang et al., 2013; Schmidt et al., 2015), likely via the activation of protein kinase C (Schmidt et al., 2015). (S)-3,5-Dihydroxyphenylglycine infusion into the NAshell also promotes cocaine seeking (Schmidt et al., 2015). Infusion of the mGluR5 antagonist MTEP into the NAcore inhibited cue- and drug-induced cocaine seeking (Knackstedt et al., 2014) and cue-induced reinstatement of ethanol seeking (Sinclair et al., 2012). These data suggest that blockade of mGluR5 prevents synaptic potentiation of MSNs in response to glutamate overflow occurring during cue- and drug-primed drug seeking (Fig. 4). Importantly, infusion of MTEP into the NAcore had no effect on cue-induced sucrose seeking (Sinclair et al., 2012), making blockade of postsynaptic mGluR5 a more attractive pharmacological approach for preventing drug seeking than the activation of presynaptic mGluR2/3 receptors (Olive, 2009). Infusion of the mGluR5 antagonist MPEP, directly into the NAshell, also reduces cocaine context-induced locomotion (Martínez-Rivera et al., 2013) as well as cocaine-primed reinstatement of cocaine seeking (Kumaresan et al., 2009). However, it is important to note that although MPEP and MTEP are both mGluR5 antagonists, studies show that MPEP may inhibit NMDARs to a certain extent, whereas MTEP has fewer off-target effects and is more selective for mGluR5 than mGluR1 compared with MPEP (Lea and Faden, 2006).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 4.

Pharmacological targets at the glutamatergic NAcore synapse. Shown here is a schematic of a glutamate synapse in the NAcore with the pre- (green) and postsynaptic (blue) terminals as well as an astrocytic contact (yellow). Glutamate is depicted as orange spheres and cysteine is shown as gray spheres. Listed next to AMPA, NMDA, mGLuR2/3, mGluR1, mGluR5, mGluR7, x_c-, and GLT-1 are the drugs that affect these proteins, which have been shown to inhibit drug seeking.

D. Group II Metabotropic Glutamate Receptors (Metabotropic Glutamate Receptors 2 and 3)

As discussed above, mGluR2/3 receptors are Gi/Go coupled and are normally localized presynaptically (Shigemoto et al., 1997); when activated, these autoreceptors act to limit synaptic release probability. Accordingly, systemic administration of an mGluR2/3-selective agonist such as LY379268 [(1S,2R,5R,6R)-2-amino-4-oxabicyclo[3.1.0]hexane-2,6-dicarboxylic acid] inhibits cue- and cocaine-induced cocaine reinstatement (Baptista et al., 2004; Peters and Kalivas, 2006; Cannella et al., 2013), cue- and context-induced nicotine reinstatement (Liechti et al., 2007), cue- and context-induced heroin reinstatement (Bossert et al., 2004, 2005), stress- and cue-induced ethanol reinstatement (Zhao et al., 2006), and cue-induced and drug-primed reinstatement of methamphetamine seeking (Kufahl et al., 2013). When examined, these studies show that systemic LY379268 administration also inhibits cue- and pellet-induced food seeking, although at a higher threshold dose than for inhibiting drug seeking.

In the accumbens, mGluR2/3 is expressed on cortical terminals synapsing on MSNs (Moran et al., 2005). Microdialysis studies show that blockade increases extracellular glutamate levels, whereas activation of mGluR2/3 has the opposite effect, supporting a role for mGluR2/3 autoreceptors in regulating glutamate release at corticoaccumbal synapses (Moussawi and Kalivas, 2010). The tonic activation of mGluR2/3 can be negatively affected by drug-induced alterations in NAcore basal glutamate levels or regulation of receptor function, making activation of this receptor system an attractive candidate for therapeutic intervention. Accordingly, activation of mGluR2/3 receptors in the accumbens proves to be an effective mechanism for inhibiting drug seeking (Table 3) (Olive, 2009). However, it is important to note that mGluR2/3 agonist microinjection into the NAcore inhibits locomotion (Besheer et al., 2010) as well as sucrose seeking (Peters and Kalivas, 2006), indicating potential complications for mGluR2/3 agonists as a therapeutic strategy for treating addiction. Direct infusion of LY379268 into the NAcore inhibits cocaine-primed reinstatement (Peters and Kalivas, 2006), cue-induced reinstatement of ethanol seeking (Besheer et al., 2010; Griffin et al., 2014), cue-induced heroin seeking (Bossert et al., 2005), and hyperlocomotion in rats previously exposed to amphetamine (Chi et al., 2006). Furthermore, systemic administration of LY379268 inhibits increased dopamine in the NAshell elicited by nicotine administration in a nicotine-paired context (D'Souza et al., 2011), supporting a possible role for hetero-mGluR2/3 receptors that presynaptically regulate dopamine release (Baker et al., 2002). In addition, infusion of LY379268 directly into the NAshell reduced cue-induced nicotine seeking (Liechti et al., 2007), as well as context-induced heroin seeking (Bossert et al., 2006).

An emerging compound showing promise in treating addiction to multiple classes of addictive substances is trifluoromethoxy)phenyl]methyl]-3H-isoindol-1-one (AZD8529), an mGluR2-specific positive allosteric modulator. Systemic administration of this compound decreases cued nicotine intake and cue- and nicotine-induced seeking (Justinova et al., 2015) as well as cue-induced methamphetamine seeking (Caprioli et al., 2015). Interestingly, AZD8529 was effective at inhibiting cued nicotine seeking at doses that did not affect food seeking, indicating that the selective activation of mGluR2 may prove a more effective treatment of relapse given the lack of a negative effect on natural rewards.

E. Group III Metabotropic Glutamate Receptors (Metabotropic Glutamate Receptor 7)

Similar to mGluR2/3 receptors, mGluR7 receptors are presynaptically localized (Li and Markou, 2015). However, in contrast with mGluR2/3 stimulation, mGluR7 activation augments glutamate and GABA release (Li et al., 2013). Systemic administration of AMN082 (N,N′-dibenzhydrylethane-1,2-diamine dihydrochloride), a selective mGluR7 agonist, inhibits cocaine intake and cocaine- and heroin-primed reinstatement (Li et al., 2010), as well as ethanol intake and ethanol-primed CPP (Salling et al., 2008; Bahi et al., 2012). Interestingly, microinjection of the mGluR7 agonist AMN082 in the accumbens increased glutamate levels, whereas it decreased GABA and had no effect on dopamine. The increase in glutamate by AMN082 appears to activate mGluR2/3, since the ability of intra-NAcore infusion of AMN082 to block cocaine-primed reinstatement was blocked with coadministration of an mGluR7 antagonist MMPIP [6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin-4(5H)-one] or with the mGluR2/3 antagonist LY341495. Mechanistically, dialysis experiments show that AMN082 blocked cocaine-mediated enhancement of NAcore glutamate in animals after self-administration and extinction, an effect that was reversed by pretreatment with LY341495 (Li et al., 2013). Taken together, these data indicate that the inhibition of drug seeking by activating mGluR7 relies on stimulating mGluR2/3, thereby inhibiting synaptic glutamate release.

F. Glial Glutamate Release and Uptake

Glutamate synaptic transmission in the accumbens is heavily regulated by extrasynaptic glutamate tone provided and maintained by astroglial cells (Scofield and Kalivas, 2014). Given the importance of glutamate transmission in the accumbens with respect to initiating drug-seeking behavior, the mechanisms of glial glutamate release and uptake have been proposed to be particularly relevant in understanding the neurobiology of relapse vulnerability (Kalivas, 2009). Chronic drug exposure alters glutamate synaptic plasticity in the accumbens in part by reducing the expression level of glial proteins that regulate homeostatic levels of extrasynaptic glutamate through glutamate release (via the glial cysteine-glutamate exchanger x_c-) and uptake (via the glia GLT-1) (Scofield and Kalivas, 2014). Drug-induced disruption of these processes affects plasticity by influencing extrasynaptic glutamate levels, leading to the activation or lack of activation of the extrasynaptic mGluRs that influence glutamatergic plasticity (discussed above). Just as pharmacological manipulation of accumbens glutamate receptor systems is an efficient method of manipulating drug-associated behaviors for multiple classes of addictive substances, accumulating evidence indicates that drug-related behaviors in rodents and humans can also be inhibited by regulating the function of these two astroglial processes: glutamate release and uptake (Scofield and Kalivas, 2014).

Ceftriaxone is a cephalosporin β-lactam antibiotic used primarily in the treatment of bacterial meningitis (Knackstedt et al., 2010a). When administered systemically, ceftriaxone enhances GLT-1 and x_c- expression and function in the NAc (Trantham-Davidson et al., 2012; Fischer et al., 2013). The fact that ceftriaxone can reverse drug-induced alterations in synaptic glutamate homeostasis makes it an attractive candidate for the treatment of addiction. Ceftriaxone works best when given repeatedly, and typical treatment regimens range from three to seven uninterrupted sequential doses at 100–200 mg/kg (Scofield and Kalivas, 2014). In animal models of addiction and relapse, ceftriaxone treatment reduces ethanol consumption in alcohol-preferring rats (Sari et al., 2013) and inhibits both cue-induced and cocaine-primed reinstatement (Knackstedt et al., 2010a; Sondheimer and Knackstedt, 2011), as well as cue-induced reinstatement of heroin seeking (Table 4) (Shen et al., 2014b). Studies also show that ceftriaxone inhibits physical dependence and abstinence-induced withdrawal to cocaine amphetamine and methamphetamine using a planaria (flatworm) model system (Rawls et al., 2008). Mechanistically, ceftriaxone-mediated inhibition of drug seeking occurs through normalizing extrasynaptic glutamate levels and by promoting glutamate uptake to countermand drug- or cue-induced glutamate overflow in the NAcore (Kalivas, 2009; Trantham-Davidson et al., 2012). Studies show that ceftriaxone enhancement of the activity and expression of GLT-1 is required for efficacy in inhibiting cued cocaine and heroin seeking (Fischer et al., 2013; Shen et al., 2014b). Perhaps the most promising aspect of ceftriaxone’s value as a therapy for addiction is that it provides a long-lasting therapeutic window, allowing protection from cocaine relapse in rodent models when administered weeks before reinstatement (Sondheimer and Knackstedt, 2011). Interestingly, clavulanic acid is a novel structural analog of ceftriaxone that retains the β-lactam core yet has negligible antibiotic activity. Clavulanic acid has greater oral availability and brain penetrability compared with ceftriaxone and enhances expression of GLT-1. Studies show that clavulanic acid treatment inhibits cocaine intake (Kim et al., 2016) as well as morphine CPP (Schroeder et al., 2014). Additional experimentation is required to determine whether clavulanic acid will surpass ceftriaxone as the most effective β-lactam–based treatment of relapse vulnerability.

Modafinil (2-diphenylmethyl-sulfinyl-2 acetamide) is a cognitive-enhancing agent commonly used for treating narcolepsy (Mahler et al., 2014a). Modafinil appears to have a variety of targets and has been reported to modulate dopamine, serotonin, glutamate, norepinephrine, orexin, and histamine systems in the brain (Gerrard and Malcolm, 2007; Mahler et al., 2014a). Because of its ability to increase extracellular dopamine levels, modafinil may serve as replacement therapy for treating psychostimulant addiction; yet paradoxically, modafinil does not induce a robust reinforcing effect in either humans or rodents (Mahler et al., 2014a). Interestingly, systemic administration of modafinil increases extracellular glutamate levels in the NAcore and inhibits cocaine-primed reinstatement. Modafinil’s effects appear to occur through activation of x_c-, and subsequent activation of mGluR2/3, because blockade of x_c- or mGluR2/3 in the NAcore inhibits the ability of systemically administered modafinil to block cocaine-primed reinstatement (Mahler et al., 2014a). Systemic administration of modafinil also inhibits context-induced, cue-induced, and methamphetamine-primed reinstatement of methamphetamine seeking (Reichel and See, 2010). In addition, systemic delivery of modafinil inhibits drug-primed reinstatement of morphine CPP, and this effect is dependent on mGluR2/3 signaling (Tahsili-Fahadan et al., 2010).

NAC is an antioxidant drug and dietary supplement that is a precursor to glutathione and is used in the treatment of acetaminophen poisoning (Murray et al., 2012b). NAC has diverse effects, including antioxidant activity, inhibition of inflammatory cytokine release, and modulation of dopamine release. Importantly, because NAC is a cystine prodrug, it also serves as a substrate for cystine-glutamate exchange and promotes glial glutamate release via activation of x_c- (Murray et al., 2012b). Moreover, like ceftriaxone, NAC restores expression of GLT-1 and the catalytic subunit of the cystine-glutamate exchanger, xCT, in animals with a history of cocaine exposure (Knackstedt et al., 2010a). Because it promotes glial glutamate release, NAC treatment is an effective means of restoring inhibitory tone on presynaptic mGluRs. Given its ability to upregulate GLT-1, NAC also limits the extent of cue- and drug-induced synaptic glutamate spillflow underlying the reinstatement of drug seeking (Kalivas, 2009). Accordingly, systemic NAC administration reduces cue-induced and drug-primed reinstatement of cocaine (Baker et al., 2003; Murray et al., 2012a; Ducret et al., 2015), heroin (Zhou and Kalivas, 2008), and nicotine seeking (Ramirez-Niño et al., 2013) and also inhibits ethanol CPP (Ferreira Seiva et al., 2009). Physiological analyses reveal that cocaine- and heroin-induced loss of LTD and LTP induced in the NAcore after in vivo electrical stimulation of the PFC is reversed by daily NAC treatment (Moussawi et al., 2011; Shen and Kalivas, 2013). Interestingly, the ability of NAC to restore plasticity at PFC synapses in the NAcore requires signaling through mGluR2/3 (Moussawi et al., 2009). Like ceftriaxone and reinstated heroin seeking discussed above, restoration of GLT-1 expression by NAC in the NAcore is required for inhibiting both cue and cocaine-primed reinstatement (Reissner et al., 2015). Another advantage of NAC as a therapy for addiction is robust efficacy independent of when the drug is administered. NAC treatment is effective in inhibiting drug seeking if given daily during self-administration, injected for 5 days during withdrawal many weeks before a reinstatement trial, or administered acutely just prior to reinstatement trial (Madayag et al., 2007; Reichel et al., 2011; Murray et al., 2012a). In rodent models, NAC treatment also facilitates extinction learning and enhances the rate of extinction of responding for both cocaine and heroin (Moussawi et al., 2011; Murray et al., 2012a). Similar to ceftriaxone, NAC appears to provide extended relapse prevention because daily administration of NAC during abstinence inhibits cocaine seeking up to 14 days after the final NAC injection (Reichel et al., 2011). It should be noted, however, that NAC has not been shown to decrease drug self-administration when administered as drug intake is ongoing, and it also does not inhibit escalation of cocaine self-administration (Ducret et al., 2015).

G. Glial Modulators

Astroctyes are also the target of pharmacological manipulation through the inhibition of other cellular processes including phosphodiesterase (PDE) activity. Ibudilast, commonly used in the treatment of asthma, inhibits PDE activity and possesses anti-inflammatory and neuroprotective effects (Rolan et al., 2009). Systemic administration of ibudilast inhibits ethanol (Bell et al., 2015) and methamphetamine intake (Snider et al., 2013), sensitization of the locomotor response to methamphetamine (Snider et al., 2012), as well as stress- and drug-primed reinstatement of methamphetamine seeking (Beardsley et al., 2010). Ibudilast also reduces morphine withdrawal and CPP, likely as a result of its ability to reduce morphine-induced dopamine release in the NAc (Rolan et al., 2009; Schwarz and Bilbo, 2013). Although ibudilast treatment has a variety of effects that could be beneficial in the pharmacological treatment of addiction, it has yet to be determined whether inhibition of PDE activity, inflammation, or neurotrophic factor release is responsible for its effects on the inhibition of drug-seeking and drug-related behaviors.

The xanthine derivative propentofylline (PPF) inhibits both PDE activity and adenosine uptake (Sweitzer et al., 2001). However, unlike ibudilast, PPF enhances expression of GLT-1 (Tawfik et al., 2006). As such, PPF is an exciting drug that combines the therapeutic action of a glial modulator with the restoration of glutamate homeostasis (discussed in section III), is augmented by exposure to drugs of abuse, and contributes heavily to relapse vulnerability (Kalivas, 2009). As expected, PPF inhibits both cued-induced and drug-primed reinstatement of cocaine seeking (Reissner et al., 2014), as well as morphine CPP (Narita et al., 2006). Interestingly, as is the case for NAC, the ability of PPF to prevent reinstatement required the reversal of cocaine-induced downregulation of GLT-1 expression (Reissner et al., 2014).

As an extension of studies using pharmacological agents that affect astrocytes, astrocyte-specific expression of designer receptors exclusively activated by designer drugs (DREADDs) in the NAcore can be achieved using glial-specific, promoter-driven, adeno-associated viral vectors. Bull et al. (2014) demonstrate that activation of Gq signaling with the hM3D DREADD in NAcore astrocytes enhances internal calcium concentration, facilitates intracranial self-stimulation, and reduces motivation to seek ethanol after 3 weeks of abstinence. Furthermore, activation of astroglial Gq-DREADD promotes glutamate release and inhibits cue-induced cocaine seeking, likely through restoration of glutamate tone on mGluR2/3 receptors in the NAcore similar to what is described above for NAC (Scofield et al., 2015).

In summary, evidence from numerous preclinical models of addiction in rats and mice support the importance of accumbens glutamate transmission in the neurobiological substrates of addiction-related behaviors and the relapse to drug seeking. The vast degree of overlap in these findings likely results from drug-induced glutamatergic dysfunction within the corticoaccumbens circuit, a shared feature of exposure to many types of addictive drugs. Interestingly, these persistent alterations in glutamatergic plasticity are the very molecular basis for the long-lasting relapse vulnerability associated with addiction. Although there is not 100% overlap in the precise molecular alterations caused by each individual drug or in the efficacy of each type of pharmacological manipulation in suppressing addiction-related behaviors, the degree of similarity regarding the efficacy of pharmacological agents discussed above clearly illustrates the value of addiction pharmacotherapies aimed at modulating glutamate synaptic plasticity in treating addictive disorders.

A. Neurotransmitter Co-Release

Although canonically thought of as purely dopaminergic input, dopamine and glutamate co-release was recently demonstrated in the VTA mesolimbic projection to the NAc (Chuhma et al., 2004; Yamaguchi et al., 2011). Individual fibers from vesicular glutamate transporter (VGlut) 2–positive dopamine neurons form both symmetrical glutamatergic synapses and asymmetric dopaminergic synapses originating from the same axon (Sulzer et al., 1998). Co-release appears to be specific for the mesolimbic pathway, because optogenetic stimulation of VGlut2 neurons evokes robust excitatory postsynaptic potentials in NAc MSNs, but similar excitatory postsynaptic potentials cannot be detected in the dorsal striatum (Stuber et al., 2010). Co-release has an important function in mediating the psychomotor effect of stimulant drugs, because both amphetamine- and cocaine-induced sensitization are significantly reduced by genetic ablation of VGlut2 from dopamine transporter–expressing neurons (Birgner et al., 2010; Hnasko et al., 2010). Interestingly, cocaine CPP is unaffected by this intervention (Hnasko et al., 2010). In contrast with the behavioral effects observed in the sensitization models, dopamine transporter neuron-specific knockout of VGlut2 increases motivation to obtain sucrose and low doses of cocaine, as well as cue-induced reinstatement of cocaine seeking (Alsiö et al., 2011). These effects can be reconciled by the fact that the specific VGlut2 deletion reduces dopaminergic signaling in the NAc. Glutamate in synaptic vesicles facilitates the packaging of monoamines by increasing the intravesicular pH, which enhances the efficacy of the vesicular monoamine exchanger and leads to increased dopamine concentration per vesicle and enhanced dopamine release (Hnasko et al., 2010).

B. Isolation and Manipulation of the Relapse Engram

Exciting new molecular tools are currently being developed that allow manipulation of neural populations activated during a particular behavior. Early experiments with this technology began as an effort to isolate specific ensembles or groups of neurons responsible for encoding memory traces. Josselyn et al. demonstrated that groups of neurons in the lateral amygdala transiently express enhanced levels of CREB after an auditory fear conditioning (Han et al., 2008; Ploski et al., 2010). These data suggested that the activated neurons could be crucial for the fear memory. This group then used HSV viral vectors to engender CREB-dependent expression of Cre recombinase in combination with Cre-dependent expression of the diphtheria toxin receptor in the lateral amygdala to specifically isolate and destroy this population. Remarkably, after infusion of the diphtheria toxin and the selective death of the CREB-expressing neurons in this region after conditioning, freezing behavior in response to the tone was significantly reduced. Additional experiments performed by Josselyn et al. illustrate that CREB-overexpressing neurons in the lateral amygdala are also important for context-associated cocaine memory using a CPP paradigm, with post-training ablation of this population sufficient to erase the contextual cocaine memory (Hsiang et al., 2014).

Others have employed c-Fos–LacZ transgenic rats in which expression of LacZ is placed under control of the promoter for the IEG c-Fos. Given that expression of c-Fos coincides with neuronal activity, only activated neurons express the LacZ transgene in the c-Fos–LacZ rat model. After the behavior of choice, the Daun02 reagent is infused intracranially into the brain region of choice, resulting in the selective inactivation of the neurons that express LacZ due to β-galactosidase–mediated processing of Daun02 to daunoribicin (Cruz et al., 2013). Similar to the methods described above, neurons activated by a discrete stimulus can be functionally silenced to assess their role in a particular behavior. Interestingly, using this technique, inactivating neurons previously activated by a cocaine-associated context in the NAshell reduced context-mediated reinstatement of cocaine-seeking behavior (Cruz et al., 2014), specifically implicating the NAshell in drug-seeking behavior precipitated by contextual cues.

An extension of this technology functions via transient, inducible expression of Cre recombinase under direction of the promoter of an activity-dependent IEG, like the activity regulated cytoskeletal-associated protein Arc or c-Fos, deemed targeted recombination in active populations (Guenthner et al., 2013; Kawashima et al., 2014). Using this system in combination with the Cre-dependent expression of a gene that allows for control of neuronal activity (e.g., DREADD receptors or channel rhodopsin), ensembles of neurons activated during discreet behavioral tasks can be permanently targeted and manipulated at a later time point. Much like the experiments discussed above, this strategy has produced exciting results. For example, recent work deciphering the role of the cortical amygdala in odor-driven behavior has shown that the after isolation of odor-related ensembles, activation of these neuronal populations in the absence of odor recapitulates responses observed previously during odor exposure (Root et al., 2014). Furthermore, using the same mouse model, deactivating fear-related neural circuits in the hippocampus inhibits freezing behavior after exposure to a fear-inducing context (Denny et al., 2014).

Given that IEG expression can be commensurate with neuronal activity and that IEG expression is observed during reinstated drug seeking in the accumbens (Hearing et al., 2008; Kufahl et al., 2009; Mahler and Aston-Jones, 2012), these new technologies will be particularly useful in decoding and manipulating the ensemble of neurons whose activity is required for initiating drug seeking in both the accumbens and in regions that send projections to the accumbens.