Name: reinstatement of drug-seeking in mice using the conditioned place preference paradigm
Uploaded: 2018-06-07T13:00:00
Description: Watch this Scientific Journal Video about Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm at JoVE.com

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 mi...

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 ...

The overall goal of this procedure is to evaluate reinstatement of drug-seeking behavior in laboratory animals considering the impact of the drug associated environmental cues and stress. This method can help answer key question in the drug addiction field, such as the role of different environmental manipulation that can modulate relapsing to drug-seeking. The main advantage of this technique is that the CPP reinstatement is considered to reflect the reactivation of the incentive motivational value of the context stimuli paired with the drug consistent in the reappearance of the approach behavior to the context.The implications of this technique extend toward the ethology of drug relapse, because it is a useful and sensitive way to assess different environmental manipulations, which are the main triggers of human relapse. New to this method will struggle because the reinstatement model is very sensitive to environmental change. Room and environment condition should stay constant during the procedure, especially with mates of the opposite sex, noises, or light conditions.Responses to the drug and stress-induced reinstatement is performed by different procedures that can affect the results. Especially for this reason, it is important to show social defeat as a determinant factor of reinstatement. To begin, handle mice for one to two minutes each three days prior to testing.Set up identical boxes, testing room, and cocaine solution, as directed in the written protocol. Every day, bring the mice to the testing room in their home cages. Allow the mice to sit undisturbed for 15 minutes, as the habituation period to all test-related noises and stimuli.Ensure the guillotine doors are removed from the cage, and begin setting up the computer. Enter the mouse's ID, and press the trial and start command. Then, place the mouse in the middle chamber of the box, and maintain minimal noise in the testing room.When the trial is complete, gather the mice back to their home cages, and save the data on the computer. Allow each mouse to access both compartments of the boxes for 15 minutes per day, for three consecutive days. On the third day, record the amount of time each mouse spends in each compartment.Next, assign half of the mice to receive the drug in one compartment and the other half in the other compartment. First, weigh the mice to determine the appropriate cocaine dosage. Then, prepare syringes with the appropriate dosage of cocaine or saline.Inject each mouse at a time intraperitoneally, and gently place it into its assigned compartment. After four hours, inject the cocaine dose, and confine the mouse to its drug-paired compartment for 30 minutes. Ensure that the guillotine doors separating the two compartments is closed.On the post-conditioning test day, bring the animals to the testing room, and allow them to habituate for 15 minutes. Remove the guillotine doors, separating the two compartments and set up the computer program. Enter the animal IDs and press the start command.Record the amount of time spent by the untreated mice in compartment of the box during the 15 minute observation period. When the observation period is over, gather the mice back into their home cages, and save the data on the computer. Each week, conduct an extinction session with all of the mice that have demonstrated a preference for the drug-paired compartment.First, remove the guillotine door from the box, and place the animal in the box for 15 minutes. When a whole group demonstrates no preference for the drug-paired compartment, repeat the test 24 hours later to confirm the extinction. For drug-induced reinstatement, bring the animals to a different room 24 hours after extinction has been confirmed.Then, inject them with half the previous dose of cocaine, and return them to their vivarium for 15 minutes. Next, take the mice into the testing room, and place them into the box for 15 minutes. Record the amount of time each mouse spends in each compartment, and save the data on the computer.To evaluate the effects of stress-induced reinstatement, take the mice to a different room 24 hours after extinction has been confirmed. Next, place an experimental mouse into a plastic cage with an aggressive opponent mouse for 10 minutes. After the antagonistic encounter, remove the experimental mice, and bring them to the testing room.Finally remove the guillotine door, and keep the mice in the box for 15 minutes. Regarding social stress, we should confirm that the animal has actually been defeated. A positive social encounter, that does not end in defeat shows protective effects, blocking the reinstatement of the place preference of cocaine.Using the CPP protocol, male mice in this study were subjected to varying environmental manipulations to measure relapse from cocaine. Priming induced reinstatement was subjected to subsequent decreasing priming doses until the priming dose was confirmed to be ineffective. As shown in this figure, priming with 12.5, 6.25, 3.125, and 1.56 milligrams per kilogram of cocaine had a significant effect on reinstatement.Mice were subjected to a social interaction, in which the experimental animals were defeated by an aggressive opponent to measure stress-induced reinstatement. Being exposed to an aggressive animal and defeated 15 minutes before the test led to reinstating previously extinguished preference. Mice that suffered repeated social defeat before the CPP procedure, demonstrated modified rewarding effects of subthreshold doses of cocaine.After the extinction of preference, socially defeated mice reinstated their preference with the priming dose of only 0.5 milligrams per kilogram of cocaine. It is important to remember that the key point of the crack addiction research is that the development of treatments that diminish craving and consequently reduce vulnerability to relapse. Other methods, like intravenous self-administration, can be performed in order to answer additional questions like compulsivity or motivation to obtain the drug.After this development, this technique paved the way for researchers in the field of the drug addiction to explore reinstatement into drug-seeking in mice. After watching this video, you should have a good understanding of how to induce relapse through different environment manipulations, such as context or drug cues, which re-establish the place preference.

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