Name: assessment of cocaine-induced behavioral sensitization and conditioned place preference in mice
Uploaded: 2016-02-18T13:00:00
Description: Watch this Scientific Journal Video about Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice at JoVE.com

The authors thank Karen Dietz and Shari Birnbaum for previous input on behavioral design considerations and Lauren Peca for help with behavioral testing. The authors also acknowledge the generous support of the Simons Foundation (Simons Foundation Autism Research Initiative grant to C.W.C.), NIDA (DA008277, DA027664, and DA030590 to C.W.C., F32DA027265 to L.N.S. and F32DA036319 to R.D.P.), the FRAXA Research Foundation and Eleanor and Miles Shore Fellowship Program (fellowship support to L.N.S.), and the John Kaneb Fellowship Program (fellowship support to M.T.).

All experimental procedures have been approved by the McLean Hospital Institutional Animal Care and Use Committee. NOTE: The following protocol describes a single approach to CPP and locomotor sensitization, many details of which differ from other successful protocols (e.g., light- vs. dark-phase testing, consecutive vs. intermittent dosing, etc.). Novices may wish to begin with these protocols, or simply use them as guides, adapting alterations from the literature based on the experimental question(s) at hand. ...

This protocol demonstrates methods for conditioned place preference and locomotor sensitization, each of which can be used by the average lab to assess aspects of drug-induced behavioral plasticity. As with most behavioral tests, there are additional worthy considerations beyond the basic protocol. First, each of these techniques can be conceived as having two phases, induction and expression. &#34;Induction&#34; covers the development of the behavior-for CPP it occurs during conditioning, and for sensitization it is the...

Research aimed at understanding drug addiction using animal models must take a variety of approaches to address each of the assorted components that obstruct treatment success, including reward/reinforcement/motivation and withdrawal and relapse, as well as the general persistence that further complicates these issues in addiction. Since rewarding experiences associated with taking a drug of abuse are thought to motivate subsequent use, studies focusing on drug-context associations may be particularly useful for understanding brain mechanisms that contribute to drug taking and seeking. One such assay, conditioned place preference (CPP) is a high-throughput method for comparing group differences in reward sensitivity. The traditional interpretation of the task involves classical, or Pavlovian, conditioning, where a conditioned stimulus (CS) is paired with an unconditioned stimulus (UCS), and after multiple pairings, the CS elicits the same behavior as the UCS (however, see39,40). Theoretically, animals learn to associate an interoceptive state (reward or aversion) with contextual cues. The relative aversive or appetitive intensity of the interoceptive state is then assessed by then determining the animal's preference for the contextual cues. The use of place conditioning to measure drug-reward associations dates back to at least 1957, to a study using morphine on rats in a Y-maze3,4. Over the past several decades, variations on this method have been widely used to study place preference and aversion in rodents to various stimuli, and it remains particularly useful in the study of associations induced by drugs of abuse. In drug-addiction research, the assay has been used to assess the rewarding properties of a number of drugs and the contribution of different brain systems and proteins to drug reward (for reviews, see5-7,44). While there are superior methods of assessing factors that contribute to drug addiction, namely drug self-administration, CPP is a simple and much more accessible approach to measuring reward function. 
Most current protocols for conditioned place preference and aversion (CPA) use an apparatus that allows rodents to have access to two distinct chambers, either via a doorway or smaller connecting chamber. Distinctions between the two chambers are often based, at a minimum, on visual and tactile cues, including wall color and floor texture, but sometimes include other elements, such as olfactory cues. "Biased" designs typically attempt to reverse a pre-existing, innate preference for one chamber over the other, such as the one that rodents generally show for a black chamber over white. "Unbiased" designs aim to create a preference to one of two chambers that were initially equally appealing by randomly counterbalancing assignment to either chamber within a group. A "balanced" design is used when animals show small preferences, but do not, as a group, favor the same chamber. Goals of this latter design are to produce 1) pre-test preference scores for the (eventual) cocaine-paired chamber that are not significantly different between experimental groups and 2) negligible preference for the cocaine-paired chamber at pre-test, either positive or negative8. The balanced design is ideal for use with the described chambers, which utilize contradicting biases for wall color (black over white) and flooring (wire over bar), resulting in a roughly equal distribution of small preferences for both the black and white sides in different animals. Balancing calculations are described in further detail below.
During conditioning, animals are exposed to a drug and quickly placed into one of these two environments for a limited time period. Exposure is typically via intraperitoneal (i.p.) or sometimes subcutaneous (s.c.) injection, although paradigms for intravenous (i.v.) self-administration9, and intracranial infusions38 in a place preference apparatus have also been developed. These pairings are complemented by non-drug (vehicle) pairings of the same length conducted in the opposite chamber, which can take place on the same day as drug pairings or on separate days. In general, when allowed to explore the apparatus after conditioning, animals will spend more time where they received a rewarding drug (i.e., one that humans and animals will voluntarily self-administer), while they will avoid a place where they were given a drug that induced illness (e.g., lithium chloride). Several studies have been dedicated to optimizing the conditions for place preference to different drugs of abuse (for review, see7). Cocaine doses (i.p.) for mice generally range from 1 to 20 mg/kg, with doses less than 5 mg/kg often used to parse high sensitivity in one group. Two or more drug pairings are typically required for adult mice10, and the length of these pairings is an important consideration. Very low doses of cocaine require an immediate and brief conditioning, likely because this method captures the most rewarding period of the exposure. Delayed or very long conditioning periods can result in no preference, or may even induce aversion11,12. Here is presented a basic method for obtaining conditioned place preference to cocaine in adult mice.
While the CPP assay is an ideal method for assessing reward-related learning and memory of drug-context associations, behavioral sensitization is arguably easier to perform and allows the assessment of changes that develop over repeated treatment. Also known as reverse-tolerance, behaviors undergoing sensitization are incrementally enhanced over repeated exposures to a particular drug of abuse, especially psychostimulants, and cross-sensitization is known to occur between some, but not all, of these drugs. One of the first assessments of cocaine-induced locomotor sensitization, in particular, in rodents was published in 197613. A number of labs have shown that sensitized locomotion is detectable long after drug cessation, depending on the original length, location and dose of exposure14-17, and the current protocol has been used to detect sensitization as long as 10 months following seven days (30 mg/kg) of cocaine treatment in mice18. The test can be performed using either photobeam or video-tracking technologies, in apparatuses of differing sizes and shapes, making it simple for many labs to perform. The robust nature, simplicity and persistence of locomotor sensitization makes its assessment an ideal part of examining basic mechanisms of long-lasting changes in drug-induced behavior. 
As is expanded upon in the discussion, an important consideration when performing the locomotor sensitization assay is whether drug is given in the home- or test-cage environment. To take advantage of the robust sensitization that occurs when drug administration occurs outside of the home cage, this protocol employs this method. However, it has been observed that when animals are not adequately habituated to a new environment before drug exposure, a novelty-induced ceiling effect occurs on Day 1, which can partially or fully mask the progressive nature of sensitization. It is likely that this represents synergistic locomotor-activating effects of the drug together with novelty, and while the mechanisms underlying such effects may be interesting, the method described is designed to reduce the role of novelty and allow the effects of the drug to be measured more independently. While it is expected this method will be useful in the assessment of other locomotor-sensitizing drugs, it has primarily evaluated its effectiveness with cocaine in C57BL/6 mice.

<table border="0" cellpadding="0" cellspacing="0" width="1293">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Cocaine Hydrochloride USP</td>
			<td style="width:127px;">Mallinckrodt Pharmaceuticals</td>
			<td style="width:119px;">0406-1520</td>
			<td style="width:832px;">Purchase and use (Schedule II controlled substance) for research purposes requires compliance and licensure according to state and federal law.&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:215px;">Conditioned Place Preference,&#160; Three Compartment Apparatus with Manual Doors and Lights for Mouse</td>
			<td style="width:127px;">Med-Associates Inc.</td>
			<td style="width:119px;">MED-CPP-MS &#38; MED-CPP-3013</td>
			<td>Our laboratory has used these boxes; however, many alternative boxes are available &#38; acceptable.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">PAS-Home Cage Activity Monitoring Photobeam Arrays</td>
			<td style="width:127px;">San Diego Instruments</td>
			<td style="width:119px;">2325-0223 &#38; 7500-0221</td>
			<td>Our lab houses these arrays inside of custom built chambers, as described in the text.&#160; There are alternatives available.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Disposable Sani-Cloth disenfecting wipes</td>
			<td style="width:127px;">PDI</td>
			<td style="width:119px;">13872</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Representative results from the CPP assay are shown in Figure 6 using wild-type C57BL/6N mice at approximately nine weeks of age. The study design was a 2 x 3 mixed factorial, with a within-subjects variable of Test (pre and post) and a between-subjects variable of Treatment (saline and cocaine 5 and 10 mg/kg). A RM ANOVA showed a significant interaction between Test and Treatment (F2,20=3.68, p&#60;0.05), which was interpreted in lieu of significant main effects observed for both Tes...

Watch this Scientific Journal Video about Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice at JoVE.com

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

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 ...

assessment-cocaine-induced-behavioral-sensitization-conditioned-place

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Behavior

Unit 1

Drug Abuse and Addiction

SCIENTISTS IN ACTION

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 Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Behavioral Assessment of Hearing in 2 to 4 Year-old Children: A Two-interval, Observer-based Procedure Using Conditioned Play-based Responses

Collecting reliable behavioral data from toddlers and preschoolers is challenging. As a result, there are significant gaps in our understanding of human auditory development for these age groups. This paper describes an observer-based procedure for measuring hearing sensitivity with a two-interval, two-alternative forced-choice paradigm. Young children are trained to perform a play-based, motor response (e.g., putting a block in a bucket) whenever they hear a target signal. An experimenter observes the child&#39;s behavior and makes a judgment about whether the signal was presented during the first or second observation interval; the experimenter is blinded to the true signal interval, so this judgment is based solely on the child&#39;s behavior. These procedures were used to test 2 to 4 year-olds (n = 33) with no known hearing problems. The signal was a 1,000 Hz warble tone presented in quiet, and the signal level was adjusted to estimate a threshold corresponding to 71%-correct detection. A valid threshold was obtained for 82% of children. These results indicate that the two-interval procedure is both feasible and reliable for use with toddlers and preschoolers. The two-interval, observer-based procedure described in this paper is a powerful tool for evaluating hearing in young children because it guards against response bias on the part of the experimenter.

This paper describes a procedure for measuring hearing sensitivity in 2 to 4 year-old children. Children are trained to perform play-based responses when they hear a target signal. Thresholds are then estimated in a two-interval, two-alternative forced-choice paradigm, based on observations of the child&#39;s behaviors.

Collecting reliable behavioral data from toddlers and preschoolers is challenging. As a result, there are significant gaps in our understanding of human ...

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.

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 ...

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 Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and ...

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...

Two Different Real-Time Place Preference Paradigms Using Optogenetics within the Ventral Tegmental Area of the Mouse

Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with behavioral testing in validated paradigms allows the measurement of behavioral consequences upon stimulation of distinct neurons in real-time with high spatial and temporal selectivity, and thus the establishment of causal relationships between neuronal activation and behavior. Here, we describe a step-by-step protocol for a real-time place preference (RT-PP) paradigm, a modified version of the classical conditioned place preference (CPP) test. The RT-PP is performed in a three-compartment apparatus and can be utilized to answer if optogenetic stimulation of a specific neuronal population is rewarding or aversive. We also describe an alternative version of the RT-PP protocol, the so-called neutral compartment preference (NCP) protocol, which can be used to confirm aversion. The two approaches are based on extensions of classical methodology originating from behavioral pharmacology and recent implementation of optogenetics within the neuroscience field. Apart from measuring place preference in real time, these setups can also give information regarding conditioned behavior. We provide easy-to-follow step-by-step protocols alongside examples of our own data and discuss important aspects to consider when applying these types of experiments.

Here we present two easy-to-follow step-by-step protocols for place preference paradigms using optogenetics in mice. Using these two different setups, preference and avoidance behaviors can be solidly assessed within the same apparatus with high spatial and temporal selectivity, and in a straightforward manner.

Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with ...

Flypub To Study Ethanol Induced Behavioral Disinhibition and Sensitization

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the underlying neurobiological mechanisms, for which diverse experimental approaches and model systems are needed. The main ingredient of alcoholic beverages is ethanol, which causes adaptive changes in the central nervous system and behavior upon chronic intake. Behavioral sensitization (i.e., escalated responses) in particular represents a key adaptive change underlying addiction. Most ethanol-induced behavioral sensitization studies in animal models have been conducted on the locomotor activating effect of ethanol. A prominent effect of ethanol is behavioral disinhibition. Behavioral sensitization to the disinhibition effect of ethanol, however, is underrepresented. To address this issue, we developed the Flypub assay that allows measuring the escalated increase in disinhibited courtship activities upon recurring ethanol exposure in Drosophila melanogaster.&#160;Here, we report the step-by-step Flypub assay including assembly of ethanol exposure chambers, setup of the assay station, criteria for fly care and collection, ethanol delivery, quantification of disinhibited courtship activities, data processing and statistical analysis. Also provided are how to troubleshoot critical steps, overcome limitations and expand its utility to assess additional ethanol-induced behaviors. The Flypub assay in combination with powerful genetic tools in Drosophila melanogaster will facilitate the task of discovering the mechanism underlying ethanol-induced behavioral sensitization.

The Flypub assay measures the behaviors that the fruit fly Drosophila melanogaster displays under the influence of ethanol. The assay can be readily mastered by experimenters at all levels and applied to various vaporized stimuli, facilitating substance abuse and addiction studies.

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the ...