**2. Animal models**

To gain an understanding of the neurobiological mechanisms underlying AUDs, numerous animal models have been developed. These models are designed to reflect various aspects of alcoholism. The most widely used procedures assess ethanol reward and reinforcement and include drinking, self-administration, and conditioned place preference (CPP). These animal models share significant homology to certain elements of AUDs in humans, such as the patterns of alcohol consumption, responses to alcohol-associated cues, and alcohol-seeking behavior. Note that, in this review, the terms reward and reinforcement are distinguished from one another. Although reward is used to refer to the appetitive nature of a stimulus as indicated by the ability of environmental stimuli to elicit approach behavior, reinforcement will refer to experimental contingencies that increase the likelihood of behavior(s) occurring [6, 7]. It should be noted that no animal model could fully emulate all aspects of human alcoholism. However, animal models allow for unparalleled access to the brain and thus provide a means to evaluate neural mechanisms involved in the aspects of alcohol reward and dependence. These models therefore represent invaluable preclinical tools for identifying potential biological correlates of and treatments for AUDs.

#### **2.1. Drinking**

depending on the treatment sought [1]. Lack of information on the neurobiological antece‐ dents and psychological determinates of relapse makes AUDs all the more problematic to

Further complicating our understanding of the persistent risk of relapse are the complex interactions between internal processes and the external environment. Most noted are the relationships that develop between environmental stimuli (cues), both contextual and discrete, and the internal states produced by alcohol. Over the course of alcohol use, specific cues become associated with the effects of alcohol through a Pavlovian learning process, whereby an associative (alcohol-cue) relationship is formed. Once the relationship has been acquired, these associative cues are able to autonomously produce psychological and physiological states that are powerful enough to elicit behavior responses. These responses have been

Even after lengthy periods of abstinence, exposure to drug-associated cues can trigger intense feelings of craving and drive drug seeking [2–5], leading to relapse to drug use. When considering alcohol in particular, this lingering sensitivity to related cues is especially prob‐ lematic given the omnipresent nature of alcohol and alcohol-related cues in society. Therefore, it is important that the neurobiology of this phenomenon be understood so that more effective

In the following sections, we describe common methodologies used to probe the neurobiology underlying primary and conditioned ethanol reward. Specifically, these methodological sections detail several commonly used animal models and tools to manipulate the brain. We then discuss the neural substrates that have been identified in ethanol-seeking behavior using

To gain an understanding of the neurobiological mechanisms underlying AUDs, numerous animal models have been developed. These models are designed to reflect various aspects of alcoholism. The most widely used procedures assess ethanol reward and reinforcement and include drinking, self-administration, and conditioned place preference (CPP). These animal models share significant homology to certain elements of AUDs in humans, such as the patterns of alcohol consumption, responses to alcohol-associated cues, and alcohol-seeking behavior. Note that, in this review, the terms reward and reinforcement are distinguished from one another. Although reward is used to refer to the appetitive nature of a stimulus as indicated by the ability of environmental stimuli to elicit approach behavior, reinforcement will refer to experimental contingencies that increase the likelihood of behavior(s) occurring [6, 7]. It should be noted that no animal model could fully emulate all aspects of human alcoholism. However, animal models allow for unparalleled access to the brain and thus provide a means to evaluate neural mechanisms involved in the aspects of alcohol reward and dependence. These models therefore represent invaluable preclinical tools for identifying potential biological correlates

suggested to play an important role in the development of AUDs and relapse.

and durable treatments for alcoholism can be designed.

48 Recent Advances in Drug Addiction Research and Clinical Applications

these models and tools.

**2. Animal models**

of and treatments for AUDs.

address.

For nearly a century, it has been known that rodents, like humans, will voluntarily consume alcohol [8, 9]. For this reason, rodents have long been used in drinking procedures that involve home-cage access to alcohol (ethanol). This represents the simplest way to gauge ethanol reward, through consumption, which is done simply by providing rodents with a bottle and measuring the amount they drink. Although alcohol is occasionally the only solution provided in drinking studies, two-bottle choice procedures are more commonly used in rodents and yield an additional measure of preference for alcohol. In a two-bottle choice drinking proce‐ dure, home-cage access to alcohol and another alcohol-free fluid (typically water) is provided continuously or at temporally controlled intervals. Evidence of ethanol reward is then indicated by the amount consumed and preference for the alcohol-containing solution over the other available fluid. Manipulations that affect alcohol consumption and/or preference but not water or total fluid intake are believed to have interfered with the rewarding properties of ethanol [10].

Although these studies have high face validity (as humans voluntarily consume alcohol orally), they are often limited by the fact that, like humans, rodents are sensitive to the aversive taste of ethanol. At higher concentrations, the aversive taste of ethanol makes it difficult for rodents to drink to the state of intoxication. Therefore, procedures requiring oral intake of ethanol may require water deprivation, slow increases in ethanol concentration, and/or the addition of a sweetener such as sucrose to the ethanol-containing solution to help rodents overcome the aversive taste [11, 12]. For instance, modified sucrose fading techniques [13] are a common strategy that has been used to achieve voluntary consumption of high concentration of ethanol in rodents. With this technique, sweeteners such as sucrose or saccharin are initially added to an ethanol solution then slowly faded out. However, this illustrates a pitfall of these proce‐ dures, which is that the underlying motivation for ethanol consumption is not always understood. For example, rodents may freely consume ethanol for the sweetened taste or for its caloric value. Therefore, it is not always evident that ethanol is being consumed in this procedure for its postabsorptive pharmacological effects. Additionally, intervention-induced decreases in alcohol intake in this procedure do not always indicate that a manipulation decreased the ethanol reward. It is possible that a reduced intake may reflect an enhancement of the pharmacological effects of ethanol, resulting in a leftward shift in the dose-response curve, which translates to an increased effect of ethanol at lower amounts. Furthermore, rodents tend to titrate their dose of ethanol consumption and often do not reach blood ethanol concentrations (BECs) of intoxication unless induced to consume greater volumes via sucrose fading or limited access to ethanol [14]. As a result, care must be taken when interpreting results in drinking studies, as the underlying reasons for decreased intake may not always be apparent.

#### **2.2. Self-administration**

In self-administration procedures, rodents must successfully perform an operant response (e.g., lever press or nose poke) to receive a small volume of an ethanol solution [15]. With this method, requisite responding is used to assess the reinforcing value of ethanol. A major advantage of self-administration procedures is that they allow for the assessment of ethanol reinforcement at distinct phases. Methodological manipulations in these procedures allow for the evaluation of the development of and enhancement in ethanol responding, including in the absence of drug (extinction) and the reemergence of responding to various environmental stimuli after responding has been extinguished (cue-, stress-, and ethanol-induced reinstate‐ ment). Thus, this procedure can be successfully used as a model aspect of alcohol seeking (rate of responding or latency to bar press for alcohol). Also, self-administration studies can be used to assess the animal's motivation to receive ethanol by increasing the difficulty of the requisite responding (a progressive ratio schedule, in which responding requirements are increased after every the delivery of reinforcer) and thus evaluating the willingness of the animal to work for an ethanol reinforcer [16]. As with humans, rodents will exert effort to obtain ethanol and this effort or seeking behavior can be reduced by administration of therapeutic agents indicated for the treatment of AUDs, such as naltrexone [17]. As such, reinstatement proce‐ dures have been shown to be highly useful for the preclinical evaluation of pharmacotherapies aimed at reducing ethanol relapse in humans (reviewed in [18]).

In addition to the rate and pattern of responding, self-administration procedures also yield an additional measure of amount of alcohol consumed. However, similar to drinking studies, the aversive taste of ethanol may be difficult to overcome in self-administration procedures. Therefore, liquid deprivation and fading strategies have also been used to establish operant responding for and consumption of ethanol. This similarly compromises straightforward interpretations of the underlying purpose for the behavior. Interpretation of studies using operant oral paradigms may also be complicated because it is difficult to distinguish between the phases of intake. As training procedures are necessary to establish ethanol self-adminis‐ tration and responding or intake serves as the primary dependent variable, it can be challeng‐ ing to separate acquisition and learning from seeking, for example.

#### **2.3. Place conditioning**

Another approach to modeling reward in rodents is the CPP procedure. With this Pavlovian (classical) conditioning procedure, a distinct environmental stimulus [conditioned stimulus (CS)] can acquire incentive salience after being paired with a motivationally significant stimulus [unconditioned stimulus (US)]. Ultimately, the previously neutral stimulus (CS) develops the ability to elicit a conditioned motivational response similar to the response elicited by the US. This mimics the ability of cues or contexts associated with alcohol (e.g., alcohol containers, odors, advertisements, and drinking establishments) to elicit craving and seeking for alcohol in humans with AUDs. In addition, drug-induced CPP can be established in humans in a laboratory setting, further validating this model [19]. As such, the CPP procedure is widely used to study the motivational properties of many abused drugs, and given its numerous benefits, the popularity of this procedure continues to grow. In the last decade alone, there has been a greater than two-fold increase in the total number of publica‐ tions reporting its use (**Figure 1**). Thus, this procedure is considered one of the most popular models of drug reward [20, 21].

advantage of self-administration procedures is that they allow for the assessment of ethanol reinforcement at distinct phases. Methodological manipulations in these procedures allow for the evaluation of the development of and enhancement in ethanol responding, including in the absence of drug (extinction) and the reemergence of responding to various environmental stimuli after responding has been extinguished (cue-, stress-, and ethanol-induced reinstate‐ ment). Thus, this procedure can be successfully used as a model aspect of alcohol seeking (rate of responding or latency to bar press for alcohol). Also, self-administration studies can be used to assess the animal's motivation to receive ethanol by increasing the difficulty of the requisite responding (a progressive ratio schedule, in which responding requirements are increased after every the delivery of reinforcer) and thus evaluating the willingness of the animal to work for an ethanol reinforcer [16]. As with humans, rodents will exert effort to obtain ethanol and this effort or seeking behavior can be reduced by administration of therapeutic agents indicated for the treatment of AUDs, such as naltrexone [17]. As such, reinstatement proce‐ dures have been shown to be highly useful for the preclinical evaluation of pharmacotherapies

In addition to the rate and pattern of responding, self-administration procedures also yield an additional measure of amount of alcohol consumed. However, similar to drinking studies, the aversive taste of ethanol may be difficult to overcome in self-administration procedures. Therefore, liquid deprivation and fading strategies have also been used to establish operant responding for and consumption of ethanol. This similarly compromises straightforward interpretations of the underlying purpose for the behavior. Interpretation of studies using operant oral paradigms may also be complicated because it is difficult to distinguish between the phases of intake. As training procedures are necessary to establish ethanol self-adminis‐ tration and responding or intake serves as the primary dependent variable, it can be challeng‐

Another approach to modeling reward in rodents is the CPP procedure. With this Pavlovian (classical) conditioning procedure, a distinct environmental stimulus [conditioned stimulus (CS)] can acquire incentive salience after being paired with a motivationally significant stimulus [unconditioned stimulus (US)]. Ultimately, the previously neutral stimulus (CS) develops the ability to elicit a conditioned motivational response similar to the response elicited by the US. This mimics the ability of cues or contexts associated with alcohol (e.g., alcohol containers, odors, advertisements, and drinking establishments) to elicit craving and seeking for alcohol in humans with AUDs. In addition, drug-induced CPP can be established in humans in a laboratory setting, further validating this model [19]. As such, the CPP procedure is widely used to study the motivational properties of many abused drugs, and given its numerous benefits, the popularity of this procedure continues to grow. In the last decade alone, there has been a greater than two-fold increase in the total number of publica‐ tions reporting its use (**Figure 1**). Thus, this procedure is considered one of the most popular

aimed at reducing ethanol relapse in humans (reviewed in [18]).

50 Recent Advances in Drug Addiction Research and Clinical Applications

ing to separate acquisition and learning from seeking, for example.

**2.3. Place conditioning**

models of drug reward [20, 21].

**Figure 1.** Annual number of CPP studies published over the last 20 years. Values were obtained from PubMed using the search term "CPP" OR "place preference" OR "place conditioning". Adapted from Tzschentke [20, 21].

In a standard ethanol CPP procedure, a discrete cue [e.g., visual or tactile stimulus; referred to as the positive CS (CS+)] presented in one spatial location is repeatedly paired with ethanol, usually administered by the investigator (i.e., noncontingently). On alternating sessions, a different stimulus not paired with ethanol [typically paired with saline, the negative CS (CS-)] is presented in a location adjacent to where the CS+ was presented. During this acquisition phase, an association develops between the CS+ and the subjective effects of ethanol (US). In the subsequent drug-free expression phase, animals are given access to the entire conditioning apparatus and thus to both cues (CS+ and CS-). When given the choice between the CS+ and CS-, animals assess the memory formed in the acquisition phase and will generally approach and maintain contact with (i.e., prefer) the CS+ when a US is rewarding. In other words, if an animal spends a greater amount of time with the ethanol-paired stimulus (CS+) in relation to the nondrug-paired stimulus (CS-), this is taken as an indication of the positive motivational effects of alcohol. Conversely, a greater amount of time spent with the saline-paired stimulus compared to the ethanol-paired stimulus would be considered conditioned place aversion (CPA) and taken to indicate a negative motivational effect of ethanol. The ability of alcohol to produce CPP or CPA depends on many factors, such as past history of ethanol exposure, route of administration, injection timing, and dose (e.g., [22–26]). A result of conditioned reward or aversion can vary by species and strain. Although conditioned ethanol responses have been reported in some strains of rat (e.g., [27–29]), studies overall have shown conflicting results ranging from lack of CPP [30] to CPA [31, 32]. However, ethanol CPP has been found in a wide array of mouse strains [33] and thus has much utility as a model of ethanol reward in this species.

Unlike self-administration, CPP does not require a lengthy training phase. In fact, in an inbred st rain of mouse (DBA/2J) commonly used in ethanol CPP, a significant place preference can be conditioned after only two ethanol-cue pairings [34–36]. Another advantage to this procedure is that it does not involve oral intake of ethanol, which is required in drinking and self-administration procedures. This is highly beneficial in cases where manipulations, such as pharmacology agents, reduce general consummatory behavior in addition to ethanol reward or reinforcement. For example, erroneous conclusions may be made when a drug with anorectic liability reduces the oral intake of ethanol. However, because CPP involves noncon‐ tingent ethanol administration, the effect of anorectic drugs on ethanol reward can be assessed more accurately [35]. The place preference paradigm also permits for the evaluation of manipulations on the various phases of learning an ethanol-paired cue that are often difficult to isolate in other procedures, and an understanding of these various phases of learning is important for understanding the progression of alcohol addiction. These phases include the acquisition, conditioned expression, extinction, and reinstatement of the conditioned effects of ethanol. Acquisition and conditioned expression of CPP are described in the paragraph above. Extinction occurs following repeated exposures to the CS+ unpaired with the US (ethanol), which results in a loss of conditioned responding as the animal learns that the CS+ and US are no longer associated and the CS+ loses its rewarding value. Reinstatement, the phase most comparable to relapse in the human condition of addiction, is the reemergence of ethanol-seeking behavior following extinction, usually elicited by a priming injection of a low dose of ethanol (US), stress, or ethanol cues before being placed within the conditioning apparatus. Specifically, when a manipulation disrupts the expression of ethanol CPP, this is generally taken to indicate that it interfered with the conditioned rewarding effect of ethanol or ethanol seeking. Indeed, because CPP can be used to gauge the conditioned rewarding or motivational value of stimuli, it also serves as an effective method to measure cue-induced ethanol-seeking behavior. On the other hand, manipulations that disrupt the development (acquisition) of CPP are thought to impact either associative learning or the primary rewarding effect of ethanol. To distinguish between these two possibilities, this procedure can also be used to assess whether the manipulation also disrupts the acquisition of other associations such as CPA induced by ethanol or other drugs (e.g., [34]). However, if a manipulation specifically affects the extinction of CPP, it is thought to impair the formation of an inhibitory memory, which relies on different neural structures than those required for the acquisition of CPP. If a manipulation impairs the reinstatement of CPP, it is thought to either impair mechanisms that allow for evaluation updating or reemergence of behavior, but manipulations could also prevent access to the original acquisition memory and manipulations must be assessed for specificity to reinstatement.

One disadvantage of the CPP procedure is that the drug is administered by the investigator and therefore delivered noncontingently. Although this may be considered an advantage given the control over the dose it provides, it reduces the face validity of this model. Unlike humans, in this procedure, rodents do not consume alcohol of their own volition. Similarly, humans do not take alcohol via intraperitoneal injections, as is used in this animal model. Moreover, this procedure does not typically involve an escalation in intake, as is usually observed in humans. Comparable to self-administration procedures, manipulations that affect locomotor activity may also nonspecifically impact CPP expression. It has been previously demonstrated that increases in activity may disrupt ethanol CPP expression, thereby obscuring its detection [37]. Hence, results obtained by manipulations that increased or decreased preference test activity must be cautiously interpreted. Despite these drawbacks, this model presents a rapid and efficient method to evaluate the primary and conditioned motivational effects of ethanol in rodents.

#### **2.4. Summary**

as pharmacology agents, reduce general consummatory behavior in addition to ethanol reward or reinforcement. For example, erroneous conclusions may be made when a drug with anorectic liability reduces the oral intake of ethanol. However, because CPP involves noncon‐ tingent ethanol administration, the effect of anorectic drugs on ethanol reward can be assessed more accurately [35]. The place preference paradigm also permits for the evaluation of manipulations on the various phases of learning an ethanol-paired cue that are often difficult to isolate in other procedures, and an understanding of these various phases of learning is important for understanding the progression of alcohol addiction. These phases include the acquisition, conditioned expression, extinction, and reinstatement of the conditioned effects of ethanol. Acquisition and conditioned expression of CPP are described in the paragraph above. Extinction occurs following repeated exposures to the CS+ unpaired with the US (ethanol), which results in a loss of conditioned responding as the animal learns that the CS+ and US are no longer associated and the CS+ loses its rewarding value. Reinstatement, the phase most comparable to relapse in the human condition of addiction, is the reemergence of ethanol-seeking behavior following extinction, usually elicited by a priming injection of a low dose of ethanol (US), stress, or ethanol cues before being placed within the conditioning apparatus. Specifically, when a manipulation disrupts the expression of ethanol CPP, this is generally taken to indicate that it interfered with the conditioned rewarding effect of ethanol or ethanol seeking. Indeed, because CPP can be used to gauge the conditioned rewarding or motivational value of stimuli, it also serves as an effective method to measure cue-induced ethanol-seeking behavior. On the other hand, manipulations that disrupt the development (acquisition) of CPP are thought to impact either associative learning or the primary rewarding effect of ethanol. To distinguish between these two possibilities, this procedure can also be used to assess whether the manipulation also disrupts the acquisition of other associations such as CPA induced by ethanol or other drugs (e.g., [34]). However, if a manipulation specifically affects the extinction of CPP, it is thought to impair the formation of an inhibitory memory, which relies on different neural structures than those required for the acquisition of CPP. If a manipulation impairs the reinstatement of CPP, it is thought to either impair mechanisms that allow for evaluation updating or reemergence of behavior, but manipulations could also prevent access to the original acquisition memory and manipulations must be

52 Recent Advances in Drug Addiction Research and Clinical Applications

One disadvantage of the CPP procedure is that the drug is administered by the investigator and therefore delivered noncontingently. Although this may be considered an advantage given the control over the dose it provides, it reduces the face validity of this model. Unlike humans, in this procedure, rodents do not consume alcohol of their own volition. Similarly, humans do not take alcohol via intraperitoneal injections, as is used in this animal model. Moreover, this procedure does not typically involve an escalation in intake, as is usually observed in humans. Comparable to self-administration procedures, manipulations that affect locomotor activity may also nonspecifically impact CPP expression. It has been previously demonstrated that increases in activity may disrupt ethanol CPP expression, thereby obscuring its detection [37]. Hence, results obtained by manipulations that increased or decreased preference test activity must be cautiously interpreted. Despite these drawbacks, this model

assessed for specificity to reinstatement.

In summary, animal models have been used extensively in alcohol research, with several models designed to study the various aspects of ethanol-mediated behavior. The most standard used procedures to model ethanol seeking are self-administration and CPP. Al‐ though it should be noted that no one procedure is able to mimic all features of human alcohol use, these models allow for the investigation of the underlying neural mechanisms involved in distinct alcohol-related behaviors. Furthermore, the paradigms described here do not encompass all models of AUDs available. Additional models include operant runway models, vapor chamber exposure, intracranial self-stimulation, and locomotor sensitization. In the following sections, several commonly used techniques to probe the neural structures and circuits involved in rodent behavior and their application to animal models of ethanol seeking are described.
