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...

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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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11.3K 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

The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden hand is touched. If the viewed and felt touches are given at the same time then this is sufficient to induce the compelling experience that the rubber hand is one's own hand. The RHI can be used to investigate exactly how the brain constructs distinct body representations for one's own body. Such representations are crucial for successful interactions with the external world. To obtain a subjective measure of the RHI, researchers typically ask participants to rate statements such as "I felt as if the rubber hand were my hand". Here we demonstrate how the crossmodal congruency task can be used to obtain an objective behavioral measure within this paradigm.
The variant of the crossmodal congruency task we employ involves the presentation of tactile targets and visual distractors. Targets and distractors are spatially congruent (i.e. same finger) on some trials and incongruent (i.e. different finger) on others. The difference in performance between incongruent and congruent trials - the crossmodal congruency effect (CCE) - indexes multisensory interactions. Importantly, the CCE is modulated both by viewing a hand as well as the synchrony of viewed and felt touch which are both crucial factors for the RHI.
The use of the crossmodal congruency task within the RHI paradigm has several advantages. It is a simple behavioral measure which can be repeated many times and which can be obtained during the illusion while participants view the artificial hand. Furthermore, this measure is not susceptible to observer and experimenter biases. The combination of the RHI paradigm with the crossmodal congruency task allows in particular for the investigation of multisensory processes which are critical for modulations of body representations as in the RHI.

We demonstrate how an objective measure can be employed in the widely employed rubber hand illusion paradigm. This measure is obtained by modifying the well-established crossmodal congruency task. This task allows the investigation of multisensory processes which are critical for modulations of body representations as in the rubber hand illusion.

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

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 ...

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 ...

Place and Response Learning in the Open-field Tower Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how &#8220;stress&#8221; might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function1. The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats2. Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.

The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for ...

Examining Recall Memory in Infancy and Early Childhood Using the Elicited Imitation Paradigm

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is commonly assessed using verbal report paradigms in adults, studying the development of this ability in preverbal infants and children proved challenging. Over the past 30 years, researchers have developed a non-verbal means of assessing recall memory known as the elicited or deferred imitation paradigm. In one variant of the procedure, participants are presented with novel three-dimensional stimuli for a brief baseline period before a researcher demonstrates a series of actions that culminate in an end- or goal-state. The participant is allowed to imitate the demonstrated actions immediately, after a delay, or both. Recall performance is then compared to baseline or to performance on novel control sequences presented at the same session; memory can be assessed for the individual target actions and the order in which they were completed. This procedure is an accepted analogue to the verbal report techniques used with adults, and it has served to establish a solid foundation of the nature of recall memory in infancy and early childhood. In addition, the elicited or deferred imitation procedure has been modified and adapted to answer questions relevant to other aspects of cognitive functioning. The broad utility and application of imitation paradigms is discussed, along with limitations of the approach and directions for future research.

The elicited imitation procedure was established to examine the development of recall memory in infancy and early childhood. This procedure has been widely used to establish a solid foundation of the nature of recall memory in infancy and early childhood.

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is ...

Using Eye Movements Recorded in the Visual World Paradigm to Explore the Online Processing of Spoken Language

In a typical eye tracking study using the visual world paradigm, participants' eye movements to objects or pictures in the visual workspace are recorded via an eye tracker as the participant produces or comprehends a spoken language describing the concurrent visual world. This paradigm has high versatility, as it can be used in a wide range of populations, including those who cannot read and/or who cannot overtly give their behavioral responses, such as preliterate children, elderly adults, and patients. More importantly, the paradigm is extremely sensitive to fine grained manipulations of the speech signal, and it can be used to study the online processing of most topics in language comprehension at multiple levels, such as the fine grained acoustic phonetic features, the properties of words, and the linguistic structures. The protocol described in this article illustrates how a typical visual world eye tracking study is conducted, with an example showing how the online processing of some semantically complex statements can be explored with the visual world paradigm.

The visual world paradigm monitors participants' eye movements in the visual workspace as they are listening to or speaking a spoken language. This paradigm can be used to investigate the online processing of a wide range of psycholinguistic questions, including semantically complex statements, such as disjunctive statements.

In a typical eye tracking study using the visual world paradigm, participants' eye movements to objects or pictures in the visual workspace are recorded ...

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 ...

Using the Visual World Paradigm to Study Sentence Comprehension in Mandarin-Speaking Children with Autism

Sentence comprehension relies on the ability to rapidly integrate different types of linguistic and non-linguistic information. However, there is currently a paucity of research exploring how preschool children with autism understand sentences using different types of cues. The mechanisms underlying sentence comprehension remains largely unclear. The present study presents a protocol to examine the sentence comprehension abilities of preschool children with autism. More specifically, a visual world paradigm of eye-tracking is used to explore the moment-to-moment sentence comprehension in the children. The paradigm has multiple advantages. First, it is sensitive to the time course of sentence comprehension and thus can provide rich information about how sentence comprehension unfolds over time. Second, it requires minimal task and communication demands, so it is ideal for testing children with autism. To further minimize the computational burden of children, the present study measures eye movements that arise as automatic responses to linguistic input rather than measuring eye movements that accompany conscious responses to spoken instructions.

We present a protocol to examine the use of morphological cues during real-time sentence comprehension by children with autism.

Sentence comprehension relies on the ability to rapidly integrate different types of linguistic and non-linguistic information. However, there is ...

Exploring Infant Sensitivity to Visual Language using Eye Tracking and the Preferential Looking Paradigm

We discuss the use of the preferential looking paradigm in eye tracking studies in order to study how infants develop, understand, and attend to the world around them. Eye tracking is a safe and non-invasive way to collect gaze data from infants, and the preferential looking paradigm is simple to design and only requires the infant to be attending to the screen. By simultaneously showing two visual stimuli that differ in one dimension, we can assess whether infants show different looking behavior for either stimulus, thus demonstrating sensitivity to that difference. The challenges in such experimental approaches are that experiments must be kept brief (no more than 10 min) and be carefully controlled such that the two stimuli differ in only one way. The interpretation of null results must also be carefully considered. In this paper, we illustrate a successful example of an infant eye tracking study with a preferential looking paradigm to discover that 6-month-olds are sensitive to linguistic cues in a signed language despite having no prior exposure to signed language, suggesting that infants possess intrinsic or innate sensitivities to these cues.

Eye tracking studies using a preferential looking paradigm can be used to study infants&#39;&#160;emerging understanding of, and attention to, their external visual world.

We discuss the use of the preferential looking paradigm in eye tracking studies in order to study how infants develop, understand, and attend to the world ...

A System for Tracking the Dynamics of Social Preference Behavior in Small Rodents

Exploring the neurobiological mechanisms of social behavior requires behavioral tests that can be applied to animal models in an unbiased and observer-independent manner. Since the beginning of the millennium, the three-chamber test has been widely used as a standard paradigm to evaluate sociability (social preference) and social novelty preference in small rodents. However, this test suffers from multiple limitations, including its dependence on spatial navigation and negligence of behavioral dynamics. Presented and validated here is a novel experimental system that offers an alternative to the three-chamber test, while also solving some of its caveats. The system requires a simple and affordable experimental apparatus and publicly available open-source analysis system, which automatically measures and analyzes multiple behavioral parameters at individual and population levels. It allows detailed analysis of the behavioral dynamics of small rodents during any social discrimination test. We demonstrate the efficiency of the system in analyzing the dynamics of social behavior during the social preference and social novelty preference tests as performed by adult male mice and rats. Moreover, we validate the ability of the system to reveal modified dynamics of social behavior in rodents following manipulations such as whisker trimming. Thus, the system allows for rigorous investigation of social behavior and dynamics in small rodent models and supports more accurate comparisons between strains, conditions, and treatments.

Described here is a novel automated experimental system that offers an alternative to the three-chamber test and also solves several caveats. This system supplies multiple behavioral parameters that enable rigorous analysis of small rodent behavioral dynamics during the social preference and social novelty preference tests.