This protocol describes the Conditioned Place Preference (CPP) as a model of relapse. This procedure permits the measurement of relapse in laboratory animals, considering the impact of drug-associated environmental cues as craving and relapse in abstaining addicts is currently the focus of drug-abuse treatment programs.
The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to acquire a conditioned place preference in a drug-paired compartment, and after the post-conditioning test, they perform several sessions to extinguish the established preference. The CPP permits the evaluation of the conditioned rewarding effects of drugs related to environmental cues. Then, the extinguished CPP can be robustly reinstated by the non-contingent administration of a priming dose of the drug, and by exposure to stressful stimuli. Both methods will be explained here. When the animal reinitiates the behavioral response, a reinstatement of the conditioned reward is considered to have taken place.
The main advantages of this protocol are that it is non-invasive, inexpensive, and simple with good validity criteria. In addition, it allows the study of different environmental manipulations, such as stress or diet, which can modulate relapse into drug seeking behaviors. However, one limitation is that if the researcher aims to explore the motivation and primary reinforcing effects of the drug, it should be complemented with self-administration procedures, as they involve operant responses of animals.
The Conditioned Place Preference (CPP) paradigm offers a simple way of assessing the conditioned reward induced by diverse stimuli1,2, and has been used broadly to study the conditioned rewarding effects of addictive drugs3. It is based on Pavlovian conditioning, evaluating the motivational value of drug-associated environmental cues for maintaining addictive behavior4. In this model, environmental cues acquire secondary appetitive properties (conditioned rewarding effects) when paired with a primary reinforcer3. For example, an initially neutral place (such as the color of one compartment in the CPP cage) is paired with the specific effects of a drug of abuse during some conditioning sessions5, while another compartment is associated with the injection of a vehicle. Following conditioning, if the animal spends more time in the compartment previously associated with the drug, it is assumed that CPP has developed3. The establishment of the preference is achieved when the animal gives a positive value to the environmental cues linked to the drug, which is the primary reinforcer. Consequently, animals will perform behavioral drug-seeking responses in response to those contextual cues6. The CPP model permits the evaluation of the rewarding properties of subthreshold doses of the drug, showing whether animals in a specific condition (e.g., having suffered from social defeat previously) are more vulnerable and sensitive to doses that are not effective in naive animals7.
The CPP model has also been used to evaluate extinction/reinstatement as an animal model to study relapse3, which is the aim of the present protocol. There are three different phases: acquisition, extinction, and reinstatement (Figure 1). In the CPP reinstatement model, animals first acquire the CPP for a drug-paired compartment, and then they perform several extinction sessions. We define extinction as the moment in which the animal reduces its behavioral responses of approximation to a conditioned rewarding stimulus that has been removed (e.g., the drug)8. During the extinction sessions, animals explore the compartments in the absence of the drug, so that the acquired preference is gradually attenuated9. An important issue to consider is that the behavioral change that the animal exhibits during extinction (the progressive decrease in the time spent in the drug-paired compartment) can be due to new learning processes that compete with the previous learned response, or due to a decrease in the internal motivational state of the subject3. Finally, the reestablishment of the place preference through the context or drug cues would be our model of reinstatement1.
Administering a priming injection of the associated drug can reinstate the preference, which is considered a reestablishment of the approximation to the contextual cues. Drug priming reinstatement occurs due to the persistent memory of the pleasurable effects of the drug, which induced craving and motivates animals to seek the environmental cues related to reward.
Some advantages of the CPP reinstatement model are that the procedure is non-invasive (in contrast with self-administration, which requires surgery), inexpensive, and simple. In addition, this model has a good criterion validity, as it mimics well what occurs in humans10,11, inducing reinstatement with stimuli that induce relapse, such as re-exposure to the drug12,13 or stress14.
There are other techniques such as the extinction – reinstatement model of intravenous self-administration. Here, animals press a lever to self-administer the drug, which permits the evaluation of the operant response of the animal, compulsivity, and motivation14,15,16. The main advantage of the CPP over self-administration procedures is that CPP reinstatement is considered to reflect the reactivation of the incentive-motivational value of the context stimuli paired with the drug, consisting of the reappearance of the approach behavior to the context17. Moreover, non-drug stimuli, such as stress, can also induce reinstatement18,19. For example, one self-administration study described no effects on reinstatement of heroin intake in rats after a foot shock or restraint stress20. Authors discussed that it was unsuccessful because those stressors were tested outside the self-administration chamber in a different context. In contrast, when using the CPP model of reinstatement, there was a clear re-establishment of morphine-induced CPP after using the same stressors, and applied in a different context to that of the CPP and at different times (0 and 15 min after stress exposure)18.
Several studies in the literature have shown different ways of drug and stress-induced reinstatement. On the one hand, drug-induced reinstatement has been reported in rats and mice using morphine5,21,22,23, cocaine24,25, amphetamine26,27, ethanol28,29, and 3,4-Methylenedioxymethamphetamine (MDMA)30. On the other hand, exposure to stress may be a determining factor in vulnerability to drug abuse. Stress is known to increase the rewarding effects of drugs7,31,32 and their role in relapse is well established33,34. For example, defeat in social interactions with a conspecific reinstates morphine and cocaine CPP18,19. In addition, animals exposed to repeated social defeat are more vulnerable to the conditioned rewarding effects of subthreshold doses of cocaine, and reinstate the preference with very low doses of cocaine7.
Application of the CPP reinstatement model is a useful and sensitive way to evaluate vulnerability to relapse in animals, and permits the assessment of different subtle environmental manipulations, which are the main triggers that threaten human relapse, such as drug- or stress-induced reinstatement.
All procedures involving mice and their care complied with national, regional, and local laws and regulations, which are in accordance with Directive 2010/63/EU of the European Parliament and the council of September 22, 2010 on the protection of animals used for scientific purposes. The Animal Use and Care Committee of the University of Valencia approved the present protocol.
1. Materials and Set-up for Conditioned Place Preference:
2. Test
3. Statistical Analysis
NOTE: Ideal sample size should be a minimum of 15 animals per group.
Firstly, representative results from the priming- and stress-induced reinstatement are shown in Figure 7 using adult OF1 male mice.
The data in Figure 7a-b representing time spent in the drug-paired compartment(s) in the Pre- and Post-C test were analyzed with a repeated measures ANOVA with a within-subjects variable Days (comparing Pre-C and Post-C). The results showed a significant effect of the variable days [F(1,34) = 51.179; p <0.001], as all the animals developed a preference in the Post-C day compared to the Pre-C. All animals were conditioned with 25 mg/kg cocaine and when extinction was achieved, one group received a priming dose of 12.5 mg/kg cocaine to test the reinstatement (Figure 7a) and the other was subjected to a social interaction in which the experimental animal was defeated by an aggressive opponent (Figure 7b). 15 min after the two events, animals were tested in their corresponding CPP box.
The priming-induced reinstatement (Figure 7a) was subjected to subsequent decreasing priming doses (half the previous one) until a priming dose is confirmed to be ineffective. The doses were: 12.5, 6.25, 3.125, and 1.56 mg/kg cocaine. The Student´s t-test revealed a significant effect on the 12.5 mg/kg cocaine reinstatement with respect to the extinction session (t statistic (t) = 20.589, degrees of freedom (d.f.) = 17; p <0.001) and subsequent doses (6.25 mg/kg: t = 18.356; 3.125 mg/kg: t = 14.260; 1.56 mg/kg: t = 16.934 d.f. = 17; p <0.001). Priming with 0.78 mg/kg did not induce the reinstatement.
Regarding the stress-induced reinstatement (see Figure 7b), being exposed to an aggressive animal and defeated 15 min before the test leads to reinstating the previously extinguished preference (t = 26.810; d.f. = 15; p <0.001)19.
Other environmental manipulations, such as suffering repeated social defeat before the CPP procedure, modify the rewarding effects of subthreshold doses of cocaine (Figure 7c). A mixed ANOVA with the between variable "stress" and the within variable "days" was performed, and there was a significant effect of the interaction Stress x Days [F(1,29) = 4.144; p <0.05]. Only socially defeated animals exhibited preference for the drug-paired compartment with a subthreshold dose of cocaine, which has been shown to be ineffective in standard animals7. In addition, after extinction of the preference, socially defeated animals reinstated their preference with only 0.5 mg/kg cocaine priming (t = 25.484 d.f. = 17; p <0.001).
Secondly, as a negative result, we can see how reinstatement can be prevented by a positive social interaction with a non-aggressive conspecific (Figure 8a). Therefore, it is important to evaluate whether animals are defeated or not after a social encounter37.
Another example of negative results that prevent reinstatement can be nutritional manipulations, such as a high-fat palatable diet intake. First, ANOVA analysis revealed an effect of the variable Days [F(1,26) = 21.527; p <.0.001], as both groups developed preference for the drug-paired compartment (p <0.01) (Figure 8b). After extinction, our results show that animals switched to the high-fat diet after the Post-C test decreased their sensitivity to drug-priming induced reinstatement, as they did not reinstate the preference with a 6.25mg/kg cocaine dose38.
Figure 1: Experimental procedure of the Conditioned Place Preference. It consists of three phases: Pre-Conditioning, Conditioning, and Post-Conditioning. There are 4 conditioning days in which the procedure is alternated each day, beginning with cocaine on days 1 and 3 and with vehicle on days 2 and 4. Then, extinction and reinstatement sessions are performed. Please click here to view a larger version of this figure.
Figure 2: Software usage sessions for Pre-C and Post-C tests where time spent in each compartment and number of entries is recorded. Please click here to view a larger version of this figure.
Figure 3: Data after the Pre-C and Post-C test. Here, record the data obtained during the test. Please click here to view a larger version of this figure.
Figure 4: Unbiased assignment of animals to the compartments for the next phase of conditioning. Take the data of the last Pre-C test day in the white and black compartments. Half the animals are assigned to receive the drug or vehicle in one compartment (black or white), and the other half in the other compartment. Exclude animals that show strong aversion or preference. Please click here to view a larger version of this figure.
Figure 5: Final assignment of Vehicle and Cocaine paired sessions. This figure shows alternation on the order of pairings. On days 1 and 3, an animal (e.g., 1.1) is first exposed to the cocaine-paired compartment (the white one) and, after four hours, is exposed to the vehicle-paired compartment (the black one). On days 2 and 4, the order of pairings is inverted, and the animal is first exposed to vehicle-paired (black) compartment and, after four hours, to the cocaine-paired (white) compartment. As this procedure is unbiased and counterbalanced, simultaneous conditioning sessions can be conducted with two animals using both compartments of the same box (e.g., animals 1.1 and 2.4, 3.1 and 1.3), which allows us to reduce time devoted to the experimental procedure. Note that these pairs of animals follow the reversed order of pairings through the conditioning phase. Please click here to view a larger version of this figure.
Figure 6: Two models of reinstatement. (a) Priming-induced reinstatement and (b) stress-induced reinstatement. (a) Animals receive a drug injection in a non-contingent room and are brought to the vivarium; 15 min later they are tested in the CPP box. (b) Animals are defeated in an agonistic encounter in a non-contingent room for 10 min and, afterwards, they are tested in the CPP box. Please click here to view a larger version of this figure.
Figure 7: Representative positive results. Priming (a) and stress-induced (b) reinstatement after 25 mg/kg Cocaine CPP. (c) Effects of social defeat on the rewarding effects of subthreshold doses of cocaine (1 mg/kg). Bars represent the mean (± SEM) time in seconds spent in the drug-paired compartment during pre-conditioning (white), post-conditioning (black), the last extinction session (light grey), and reinstatement (dark grey). The reinstatement test was evaluated 15 min after a priming dose of half the previous dose received. *p <0.05; **p <0.01; ***p <0.001 significant difference vs Pre-C (ANOVA with a within-subjects variable Days (comparing Pre-C and Post-C) or the previous extinction session (Student's t test). These figures have been modified from 7,19. Please click here to view a larger version of this figure.
Figure 8: Representative negative results. Effects of protective environmental manipulations on the rewarding effects of cocaine. (a) Effects of a positive social interaction before the reinstatement test. (b) Effects of a high-fat diet during the whole extinction period and reinstatement. The control group was fed with a standard diet (13% of kcal from fat, 67% kcal from carbohydrates, and 20% kcal from protein; 2.9 total kcal/g) and the high-fat diet group (45% kcal from fat, 36% kcal from carbohydrates, and 19% kcal from protein; 4.6 total kcal/g). Bars represent the mean (± SEM) time in seconds spent in the drug-paired compartment during pre-conditioning (white), post-conditioning (black), the last extinction session (light grey), and reinstatement (dark grey). The reinstatement test was evaluated 15 min after a priming dose of half the previous received dose. *p<0.05; **p<0.01; significant difference vs Pre-C (ANOVA with a within-subjects variable Days (comparing Pre-C and Post-C) or the previous extinction session (Student's t test). These figures have been modified from 37,38. Please click here to view a larger version of this figure.
The key point of drug addiction research is the development of treatments that diminish craving and, consequently, reduce the vulnerability to relapse. Thanks to the reinstatement model of the CPP paradigm, it is possible to study the influence of different procedural and environmental factors that modulate relapse, which is a priority of future research. There are some important points to consider, as the CPP paradigm is a test that is highly sensitive to environmental factors.
Modifications and troubleshooting: The CPP version of the reinstatement model should be modified depending on the drug of interest. There are different factors that may influence the magnitude of conditioned preference, such as duration and number of conditioning sessions. The number of associations usually ranges from one to six, with four being the most common. The duration of the conditioning tests is established according to the characteristics of the drug and its pharmacokinetics.
CPP studies usually involve many trials, so it is useful to use protocols that reduce the time required. One way to achieve this is to reduce the number of trials and another way is to perform two sessions of conditioning per day, as is shown in the present protocol. In this case, it is necessary to ensure that the effects of the drug from the previous session do not drag on to this second session39, which is why the interval of four hours between the cocaine – vehicle session was chosen. For example, the timing of the pairing in the case of cocaine during the conditioning phase should be every 24 h for 4 days with a pairing duration of 30 min. On the contrary, the time course of the conditioning procedure of ethanol should be every 48 h, a total of 8 days, 1 conditioning session per day, and alternating vehicle and EtOH, finally having a total of 4 EtOH pairings. Ethanol pairing should have a short duration, such as 5 min, as more time could produce aversive states. Effective doses range from 1 – 2 g/kg and subthreshold doses are 0.75 g/kg40. Priming-induced reinstatement should be half the previous dose received, and the apparatus should be the same.
However, choosing to do 1 or 2 sessions a day can affect the results in some special drugs of abuse. For example, in the case of MDMA-induced CPL with the two-session protocol, the conditioned preference in post-conditioning is not observed, whereas this can be obtained by performing a single association each day30, so MDMA should also be administered every 48 h, but duration of the conditioning session should be 30 min.
There are two main methods of extinction that could be run after the establishment of CPP, either daily injections of vehicle confining the animal in both the previously drug and vehicle conditioned compartments41,42,43, or by repeating the CPP test, letting animals explore the three compartments without the guillotine until preference is no longer observed7,18,30,38. In the present protocol, we employed the latter, because it is a type of spontaneous extinction without any pharmacological or behavioral manipulation related to conditioning (more similar to that occurring in human addicts), but both methods are acceptable and valid, as literature has shown for years.
Limitations of the technique: The most important limitation of the CPP paradigm and the reinstatement model is that they are very sensitive to any environmental change. Therefore, room and vivarium conditions should stay stable and constant during the whole procedure. Special care should be taken to aspects such as living in the same room with mates of the opposite sex, cages with artifacts like wheels that make a specific and constant noise, or light conditions. In addition to controlling these variables, the role of the researcher is also critical, as they can induce an increase in animals' stress levels and interfere with the results.
Significance with respect to existing methods: Regarding other methods, in the CPP version of the reinstatement model, the researcher is the one who administers the drug to the animal, in contrast with self-administration paradigms that evaluate the animal´s motivation to take the drug. It is important that we take this into account. This means that we are not evaluating motivation through an operant response nor the primary reinforcing effects of the drug. As we mentioned, CPP is a useful model to assess the critical role that environmental cues play on relapse in substance abuse disorders, which are in fact the main factors that modulate relapse in humans.
Future applications: Future applications of the CPP reinstatement model should be oriented to the so-called incubation of craving, which consists of a progressive increase in cue reactivity after abstinence of drugs, mimicking craving in humans. Nowadays, little is known about this in the CPP paradigm. A few years ago, only one study suggested that CPP could also measure this incubation period44. More recently, it was assessed whether or not environmental cues paired to morphine in the CPP could increase the behavioral response over the withdrawal period45. This study found that the number of entries to the CPP compartments were increasing over time, reflecting the incubation of craving and suggesting that reward memories and drug craving could be distinguished in the CPP paradigm.
Critical steps: There are several critical steps in the protocol. In order to affirm that animals reinstated their preference, extinction data should reflect a real extinction of the preference, being confirmed before the reinstatement test. Data from the extinction test should not show differences with respect to the Pre-C test but with respect to the Post-C test.
Regarding the social stress-induced reinstatement version, we should have confirmed that the animal has been actually defeated, since a positive social encounter that does not end in defeat shows protective effects blocking the reinstatement of the place preference of cocaine. If the experimental animals have not been defeated, they will be excluded from the study. We recommend recording and evaluating the agonistic encounter, measuring the latency of the aggressive behavior or submission behavior, the number of attacks, and the time spent in each of those behaviors. We consider that the experimental animal is defeated when it receives a minimum of 2 attacks (bites) and shows signs of submission (as illustrated in Figure 6). In all cases, the encounter lasts 10 min.
The authors have nothing to disclose.
Generalitat Valenciana, PROMETEOII / 2014/063; Ministry of Health, Social Services and Equality. Delegation of the Government for the National Plan on Drugs, Research Projects on Drug Dependencies, 2014I007; Ministry of Economy and Competitiveness (MINECO), Health Institute Carlos III, Network of Addictive Disorders (RTA) RD12 / 0028/0005 and RD16 / 0017/0007 and European Union, FEDER Funds " una manera de hacer Europa", and PSI2014- 51847-R.
MONPRE 2Z software | CIBERTEC S.A., Spain | N/A | |
Identical Plexiglas boxes with two equal sized compartments separated by a grey central area. | CIBERTEC S.A., Spain | N/A | The compartments have different colored walls (black vs white) and distinct floor textures (fine grid in the black compartment and wide grid in the white one). All boxes are equipped with four infrared light beams in each compartment of the box and six in the central area which allow the recording of the position of the animal and its crossings from one compartment to the other. |
Cocaine hydrochloride | Laboratorios Alcaliber S.A., Madrid, Spain | N/A | |
Animals: mice of the OF1 outbred strain | Charles River, Barcelona Spain | N/A | Male mice who arrive to the laboratory on PND 42 and on PND 60 perform the CPP test. These animals are specially indicated for social defeat and aggression models. |
Standard Diet- Teklad Global Diet 2014 | Supplied by Harlan Laboratories Models, S. L. (Barcelona, Spain) | 13 kcal % fat, 67 kcal % carbohydrates and 20% kcal protein; 2.9 kcal/g | |
High fat diet TD.06415 | 45 kcal % fat, 36 kcal % carbohydrates and 19% kcal protein; 4.6 kcal/g |