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 &#34; una manera de hacer Europa&#34;, and PSI2014- 51847-R.

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:
<ol>
	<li>Handle mice 3 days prior to testing for 1 - 2 min each. The base of the tail should be grasped.&#160;The body should be supported if possible.&#160;This is done to minimize pain and distress when handling an animal and&#160;to prevent interfering variables, such as stress. 
	NOTE: OF&#160;1 male mice 60 days old (young adults) weighing 35 - 40 g were employed in the present experiments.&#160;Drug-induced reinstatement can be performed in mice of both sexes. However, regarding stress-induced reinstatement, the social defeat paradigm is designed only for male rodents, as females do no induce an aggressive response in the resident mice.</li>
	<li>For place conditioning, use identical boxes made with two identical compartments (30.7 cm long &#215; 31.5 cm wide &#215; 34.5 cm high) separated by a smaller central grey area (13.8 long &#215; 31.5 cm wide &#215; 34.5 high). 
	NOTE: Compartments have different floor textures and wall colors (a smooth floor in the black compartment and a rough floor in the white one). Each compartment of the CPP box contains four infrared light beams and six in the central compartment. This allows the recording of the crossings between compartments and the position of the animal.</li>
	<li>Set up the room lights (off or lowered) each day. Use red lights (recommended), as animals do not perceive it and it is attenuated for the experimenter (approximately 40 lux measured at 1 m above floor level).</li>
	<li>Set up the program to register 15 min for Pre- and Post- conditioning test (see the Table of Materials for software) (Figure 2)</li>
	<li>Prepare the cocaine solution required for the whole experiment. Dissolve cocaine hydrochloride in 0.9% NaCl (saline) in a volume of 0.1 mL/10 g body weight. Vortex mixture and store at 4 &#176;C at the end of the day. 
	NOTE: Cocaine concentrations may vary depending on the study&#39;s purpose. If the aim is to evaluate the subthreshold doses of cocaine, a 1 mg/kg concentration could be used, which is ineffective in control animals, as seen in previous studies24,35. If an effective dose is the purpose, a 6 mg/kg concentration can be chosen, which is effective but does not induce reinstatement24, or a 25mg/kg concentration, which is effective and induces reinstatement of the preference19.</li>
	<li>Test the animals during their dark phase and use the same box for each animal across days.</li>
	<li>Bring animals every day to the testing room in their home cages and leave them undisturbed for 15 min as a habituation period to all the test-related noises.</li>
</ol>
2. Test
<ol>
	<li>Acquisition 
	NOTE: The procedure of Place Conditioning, unbiased in terms of initial spontaneous preference, consists of three phases: Pre-Conditioning, Conditioning, and Post-Conditioning (Figure 1).
	<ol>
		<li>Pre-Conditioning (Pre-C) (3 days) (Figure 2, Figure 3, Figure 4) 
		NOTE: Here, the neutral value of the compartments in the CPP box is evaluated. Measure the time spent in each compartment and later compare it with that spent in the same compartment in the Post-Conditioning test.
		<ol>
			<li>Bring the animals to the test room and leave them habituated for 15 min.</li>
			<li>Check that guillotines are removed from the cage.</li>
			<li>Set up the computer and the program. Enter animal IDs and press the &#34;Trial&#34; and &#34;Start&#34; command (Figure 2).</li>
			<li>Place the mouse gently into the middle chamber (grey compartment) and leave the testing room with minimum noise.</li>
			<li>Bring all the animals back into their home cages and save the data when the trial is finished.</li>
			<li>Allow mice to access both compartments of the apparatus for 15 min (900 s) per day on 3 consecutive days.</li>
			<li>On day 3, write down the time spent in each compartment over a 900 s period, and save it for the assignment of compartments in the next phase (Figure 3).</li>
			<li>Assign half the animals in each group to receive the drug or vehicle in one compartment (e.g., black), and the other half in the other compartment (e.g., white) (Figure 4). 
			NOTE: The final distribution of the animals must ensure that half of the animals are assigned to the initially preferred compartment and the other half to the non-preferred compartment. In addition, half of the animals received the drug in one compartment (for example, the black one) and the other half in the other (in this case, the white one).</li>
			<li>Balance scores in the group within both compartments as well as across groups (test group differences with an analysis of variance (ANOVA)). As approximate guidance, values should be around 360 - 370 s in the Pre-C test (Figure 4). NOTE: no significant differences should be detected between the time spent in the drug-paired and vehicle-paired compartments during the Pre-C test.</li>
			<li>Exclude animals that exhibit a strong aversion or preference for any compartment.Consider aversion as spending less time than 33% in the compartment, and consider preference when the animal spent more than 67% of the total time in that compartment.</li>
		</ol>
		</li>
		<li>Conditioning (4 days) (Figure 5)
		<ol>
			<li>Weigh the animals and use these weights for cocaine doses during conditioning.</li>
			<li>Prepare syringes with cocaine or saline based on body weights.</li>
			<li>Inject one mouse at a time intraperitoneally (ip) and immediately place it gently into the assigned black or white compartment of their box.</li>
			<li>After an interval of 4 h, inject the drug dose immediately before the mouse is confined to the drug-paired compartment for 30 min (Figure 5).</li>
			<li>Alternate this procedure each day, beginning with cocaine on days 1 and 3, and saline on days 2 and 4.</li>
			<li>Check that confinement is carried out in both cases by closing the guillotine door that separates the two compartments, making the central area inaccessible. 
			NOTE: In this phase, there is no need to set up the PC program.</li>
		</ol>
		</li>
		<li>Post-Conditioning (Post-C) (1 day)
		<ol>
			<li>Bring the animals to the test room and leave them habituated for 15 min.</li>
			<li>Remove the guillotine door separating the two compartments.</li>
			<li>Set up the computer and the program. Enter animal IDs and press the &#34;start&#34; command.</li>
			<li>Record the time spent by the untreated mice in each compartment during a 900 s observation period.</li>
			<li>When the trial is finished, bring all the animals back into their home cages and save the data.</li>
			<li>Consider that the drug has induced place preference if the time spent in the drug-paired compartment during the Post-C is significantly greater than in the Pre-C test. If the opposite occurs, consider an aversion for the drug-paired compartment.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Extinction
	<ol>
		<li>Conduct a weekly extinction session in all groups/animals that developed a preference for the drug-paired compartment.</li>
		<li>Place the animal in the apparatus (without guillotines) for 15 min (same as the Pre- and Post-C tests) (Figure 1). 
		NOTE: If the dose employed in the CPP is high (25 mg/kg), perform 1 extinction session per week. If a subthreshold dose is used (1 mg/kg), then perform 2 extinction sessions per week (e.g. Monday and Thursday).</li>
		<li>Consider the preference extinguished when data from the extinction test do not show differences with respect to the Pre-C test, but they do show differences with respect to the Post-C test.</li>
		<li>Repeat the test 24 h later in order to confirm the extinction.</li>
	</ol>
	</li>
	<li>Reinstatement of CPP
	<ol>
		<li>Priming-induced reinstatement
		<ol>
			<li>Evaluate the effects of a priming dose of cocaine 24 h after extinction has been confirmed. 
			NOTE: Priming doses are half the previous dose that the animal has received during conditioning. The reinstatement test undergoes the same protocol as in Pre-C and Post-C tests, except that animals receive the dose of cocaine in a non-contingent place 15 min before the test. The efficacy of drug priming in inducing reinstatement can be enhanced after repeated extinction/reinstatement experiences23, although the order of the doses seems not to play a role in the results obtained36.</li>
			<li>Bring the animals to a different room from the testing room (non-contingent place) (Figure 6).</li>
			<li>Inject them with half the previous dose of cocaine (e.g., if animals were conditioned with 25 mg/kg cocaine, a priming dose would be 12.5 mg/kg).</li>
			<li>Bring them back to their vivarium for 15 min.</li>
			<li>Take them into the testing room and place the animals in the apparatus immediately (without the guillotine doors separating the compartments) for 15 min (same as the Pre- and Post-C tests).</li>
			<li>Consider the difference in seconds between the time spent in the drug-paired compartment during the last Extinction session and the Reinstatement test as a measure of the degree of reinstatement of the preference induced by the drug.</li>
			<li>Repeat the extinction-reinstatement procedure every time with decreasing doses until it is confirmed to be ineffective. 
			NOTE: A saline injection control can be used to control the capacity of cocaine priming (and not of injection itself) to induce reinstatement.</li>
		</ol>
		</li>
		<li>Stress-induced reinstatement
		<ol>
			<li>Evaluate the effects of a stress-induced reinstatement 24 h after extinction has been confirmed 
			NOTE: Reinstatement tests are the same as those carried out in Post-C (free ambulation for 15 min), except that animals are tested 15 min after the social defeat in an agonistic encounter (Figure 6)</li>
			<li>Take the animals to a different room from the CPP room.</li>
			<li>Place the experimental mouse with an aggressive opponent sex matched of equal age and body weight in the plastic cage (Figure 6). 
			NOTE: The agonistic encounter consists of a 10 min test in a neutral transparent plastic cage (23x13.5x13cm) with a defeat result for the experimental mouse. To prepare the aggressive opponent, first isolate different animals that match in age and body weight as the experimental mice for 3 weeks. Train them to acquire fighting experience and test them to have high levels of aggressive territorial behavior. To obtain aggressive conspecifics, mice must be isolated for at least 3 weeks, and then trained in aggressive and attack behaviors. Animals are trained in a neutral environment, facing them in pairs. We let them bite the opponent 2 or 3 times and get them out of the cage, always ending the training before one of the two mice shows submission, so that the experience of the encounter reinforces aggressive behavior. After suffering aggression (threat and attack from the aggressive conspecific), experimental mice should present avoidance/flee and defensive/submissive behavior. The criterion is to adopt a specific posture of defeat, characterized by an upright submissive position, limp forepaws, upwardly angled head, and retracted ears (Figure 6).&#160;According to Burke et al., 2016, we consider that the social encounter is terminated earlier if the intruder displays a submissive supine posture for &#62;8 s&#160;or if 13 attack bites occurred. Another option to screen animals without risk of significant injury could be to pace the animal in a mesh enclosure with a hole, not large enough for either animal to pass.&#160; Aggression could be detected without subjecting animals to direct aggressive behavior.&#160; If an animal sustains an injury with a wound 1cm in diameter and/or&#160; direct exposure of muscle, it is excluded from the study and should be euthanized.</li>
			<li>After 10 min of agonistic encounter, take the mice into the testing room and place the animals in the CPP apparatus immediately (without the guillotine doors separating the compartments) for 15 min (same as the Pre- and Post-C tests).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Statistical Analysis
NOTE: Ideal sample size should be a minimum of 15 animals per group.
<ol>
	<li>Analyze the mean time spent in the drug-paired compartment of the group by means of a mixed ANOVA with one between variable, treatment, with two levels (Control group, Experimental group, example of two groups is in Figure 7c), and one within variable &#8211; Days, with 2 levels (Pre-C and Post-C). 
	NOTE: In the control group, animals receive the drug but have no environmental manipulations. On the other hand, in the experimental group animals are exposed to different environmental conditions. Compare the time spent in the drug-paired compartment on the Post-C day with respect the Pre-C test to see if there are any significant differences in sensitivity to the drug.</li>
	<li>In addition, perform Bonferroni post-hoc analyses to check either of the significant scores.</li>
	<li>Analyze data related to extinction and reinstatement values in the groups that developed preference by means of Student&#180;s t-tests to continue with the extinction-reinstatement procedure during the course of the experiment, but at the final moment, analyze them with an ANOVA with a within variable (Days) with the number of levels depending on each group. 
	NOTE: When the number of comparisons is increased, the number of those that will be significant by chance also increased. As such, to avoid getting false-positives (Type 1 Errors) we &#39;correct&#39; the p-value thereby making the test more conservative with the Bonferroni correction for multiple t-tests. However, for the reinstatement test, the comparison should be made between the reinstatement test and the last extinction test. Therefore, there is no need for correction.</li>
</ol>

The authors have nothing to disclose.

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.
Modifi...

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 &#8211; 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.

<table><tbody><tr><td>MONPRE 2Z software</td><td>CIBERTEC S.A., Spain</td><td>N/A</td><td></td></tr><tr><td>Identical Plexiglas boxes with two equal sized compartments separated by a grey central area.</td><td>CIBERTEC S.A., Spain</td><td>N/A</td><td>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.</td></tr><tr><td>Cocaine hydrochloride</td><td>Laboratorios Alcaliber S.A., Madrid, Spain</td><td>N/A</td><td></td></tr><tr><td>Animals: mice of the OF1 outbred strain</td><td>Charles River, Barcelona Spain</td><td>N/A</td><td>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.</td></tr><tr><td>Standard Diet- Teklad Global Diet 2014</td><td>Supplied by Harlan Laboratories Models, S. L. (Barcelona, Spain)</td><td></td><td>13 kcal % fat, 67 kcal % carbohydrates and 20% kcal protein; 2.9 kcal/g</td></tr><tr><td>High fat diet TD.06415</td><td></td><td></td><td>45 kcal % fat, 36 kcal % carbohydrates and 19% kcal protein; 4.6 kcal/g</td></tr></tbody></table>

reinstatement of drug-seeking in mice using the conditioned place preference paradigm

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.

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). T...

Watch this Scientific Journal Video about Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm at JoVE.com

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

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 ...

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11.4K Views.  Universitat de València. 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.

Video: Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats 

The most insidious aspect of drug addiction is the high propensity for relapse. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the neurobiology and phenomenology of relapse to drug use. Although procedural variations have emerged over the past several decades, the most conventional reinstatement procedures are based on the drug self-administration (SA) model. In this model, an animal is trained to perform an operant response to obtain drug. Subsequently, the behavior is extinguished by withholding response-contingent reinforcement. Reinstatement of drug seeking is then triggered by a discrete event, such as an injection of the training drug, re-exposure to drug-associated cues, or exposure to a stressor 1.
Reinstatement procedures were originally developed to study the ability of acute non-contingent exposure to the training drug to reinstate drug seeking in rats and monkeys 1, 2. Reinstatement procedures have since been modified to study the role of environmental stimuli, including drug-associated cues and exposure to various forms of stress, in relapse to drug seeking 1, 3, 4.
Over the past 15 years, a major focus of the reinstatement literature has been on the role of stress in drug relapse. One of the most commonly used forms of stress for studying this relationship is acute exposures to mild, intermittent, electric footshocks. The ability of footshock stress to induce reinstatement of drug seeking was originally demonstrated by Shaham and colleagues (1995) in rats with a history of intravenous heroin SA5. Subsequently, the effect was generalized to rats with histories of intravenous cocaine, methamphetamine, and nicotine SA, as well as oral ethanol SA 3, 6.
Although footshock-induced reinstatement of drug seeking can be achieved reliably and robustly, it is an effect that tends to be sensitive to certain parametrical variables. These include the arrangement of extinction and reinstatement test sessions, the intensity and duration of footshock stress, and the presence of drug-associated cues during extinction and testing for reinstatement. Here we present a protocol for footshock-induced reinstatement of cocaine seeking that we have used with consistent success to study the relationship between stress and cocaine seeking.

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

Neuroscience

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration 12,16. 
Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)17-22. Auditory and visual stimuli are self-administered by several species18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement24. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS24. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation 25 is not observed in this model24. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose26. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. 
In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...

A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with drug-taking. This model has been implemented in experiments using different types of substances of abuse such as methamphetamine, cocaine, and alcohol. However, punishment-induced abstinence in heroin-trained animals has not been demonstrated. Furthermore, acute stress is a key trigger for relapse in humans and animal models. It was previously demonstrated that acute food deprivation robustly induced reinstatement of extinguished cocaine and heroin seeking. The procedure described here can be used to assess the effects of acute stress exposure on heroin seeking after punishment-imposed abstinence. A total of 8 rats were implanted with chronic intravenous (i.v.) catheters and trained to self-administer heroin (0.1 mg/kg/infusion) for 18 days under a seek-take chained schedule. Completing the seek link gave access to the take lever, which was paired with a heroin infusion. The seek lever was programmed with a variable interval 60 schedule of reinforcement (VI60), and the take lever was programmed with a fixed-ratio 1 reinforcement schedule (FR1). Following self-administration training, a mild foot shock was delivered on 30% of the completed seek links instead of the extension of the take lever. Footshock intensity was increased by 0.1 mA per daily session from 0.2 mA to 1.0 mA. Heroin-seeking tests were performed after 24 h of food deprivation (FD) or sated conditions. Rats under acute food deprivation condition robustly increased heroin seeking after punishment-imposed abstinence.

A procedure that demonstrates a robust acute food deprivation-induced relapse to heroin seeking after punishment-imposed abstinence is described. A punishment-imposed abstinence model was successfully implemented using the seek and take chain schedule for heroin self-administration. Heroin-seeking tests are then performed following 24 h of food-deprivation stress.

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities.&#160;The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats

This protocol demonstrates the steps needed to use optogenetic tools to reverse cocaine-induced plasticity at thalamo-amygdala circuits to reduce subsequent cocaine seeking behaviors in the rat. In our research, we had found that when rats self-administer intravenous cocaine paired with an audiovisual cue, synapses formed at inputs from the medial geniculate nucleus of the thalamus (MGN) onto principal neurons of the lateral amygdala (LA) become stronger as the cue-cocaine association is learned. We hypothesized that reversal of the cocaine-induced plasticity at these synapses would reduce cue-motivated cocaine seeking behavior. In order to accomplish this type of neuromodulation in vivo, we wanted to induce synaptic long-term depression (LTD), which decreases the strength of MGN-LA synapses. To this end, we used optogenetics, which allows neuromodulation of brain circuits using light. The excitatory opsin oChiEF was expressed on presynaptic MGN terminals in the LA by infusing an AAV containing oChiEF into the MGN. Optical fibers were then implanted in the LA and 473 nm laser light was pulsed at a frequency of 1 Hz for 15 minutes to induce LTD and reverse cocaine induced plasticity. This manipulation produces a long-lasting reduction in the ability of cues associated with cocaine to induce drug seeking actions.

The methods described here outline a procedure used to optogenetically reverse cocaine-induced plasticity in a behaviorally-relevant circuit in rats. Sustained low-frequency optical stimulation of thalamo-amygdala synapses induces long-term depression (LTD). In vivo optogenetically-induced LTD in cocaine-experienced rats resulted in the subsequent attenuation of cue-motivated drug seeking.

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

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Chapters in this video

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

Operant Sensation Seeking in the Mouse

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

A General Method for Evaluating Incubation of Sucrose Craving in Rats

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats