Summary

Evaluatie van cocaïne geïnduceerde sensitisatie en geconditioneerd Place Preference in Muizen

Published: February 18, 2016
doi:

Summary

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.

Abstract

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.

Introduction

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.

Protocol

Alle experimentele procedures zijn goedgekeurd door de McLean Hospital Institutional Animal Care en gebruik Comite. Opmerking: Het volgende protocol beschrijft een benadering van CPP en bewegingsapparaat sensibilisatie, veel details van die verschillen van andere succesvolle protocollen (bijvoorbeeld licht- donker versus-fase test, opeenvolgende versus intermitterende dosering, etc.). Beginners kunnen wensen om te beginnen met deze protocollen, of gewoon gebruik maken van hen als gidsen, het aanpassen van wijzi…

Representative Results

Representatieve resultaten van de CPP assay worden getoond in figuur 6 gebruikt wildtype C57BL / 6N muizen ongeveer negen weken. De opzet van het onderzoek was een 2 x 3 gemengd faculteit, met een within-subjects variabele Test (pre en post) en een tussen-subjects variabele van de behandeling (zoutoplossing en cocaïne 5 en 10 mg / kg). Een RM ANOVA toonde een significante interactie tussen test en behandeling (F 2,20 = 3,68, p <0,05), die in plaats van significante hoofdeffecten …

Discussion

Dit protocol toont werkwijzen voor geconditioneerde plaatsvoorkeur en bewegingsapparaat sensibilisatie, die elk de gemiddelde lab kan worden gebruikt om aspecten van geneesmiddelgeïnduceerde behavioral plasticiteit beoordelen. Zoals bij de meeste gedragstesten, er aanvullende overwegingen verdient, naast de normale protocol. Eerste, elk van deze technieken kunnen worden opgevat als met twee fasen, inductie en expressie. "Inductie" omvat de ontwikkeling van het gedrag-van CPP dit tijdens conditioneren en het s…

Disclosures

The authors have nothing to disclose.

Acknowledgements

De auteurs danken Karen Dietz en Shari Birnbaum voor eerdere ingang op gedrags- ontwerp overwegingen en Lauren Peca voor hulp bij gedrags-testen. De auteurs ook de genereuze steun van de Simons Foundation (Simons Foundation Autism Research Initiative subsidie ​​aan CWC), NIDA (DA008277, DA027664 en DA030590 om CWC, F32DA027265 tot LNS en F32DA036319 RDP), de FRAXA Research Foundation en Eleanor en Miles erkennen Shore Fellowship Program (fellowship steun aan LNS), en de John Kaneb Fellowship Program (fellowship ondersteuning van MT).

Materials

Cocaine Hydrochloride USP Mallinckrodt Pharmaceuticals 0406-1520 Purchase and use (Schedule II controlled substance) for research purposes requires compliance and licensure according to state and federal law. 
Conditioned Place Preference,  Three Compartment Apparatus with Manual Doors and Lights for Mouse Med-Associates Inc. MED-CPP-MS & MED-CPP-3013 Our laboratory has used these boxes; however, many alternative boxes are available & acceptable.
PAS-Home Cage Activity Monitoring Photobeam Arrays San Diego Instruments 2325-0223 & 7500-0221 Our lab houses these arrays inside of custom built chambers, as described in the text.  There are alternatives available.
Disposable Sani-Cloth disenfecting wipes PDI 13872

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Cite This Article
Smith, L. N., Penrod, R. D., Taniguchi, M., Cowan, C. W. Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice. J. Vis. Exp. (108), e53107, doi:10.3791/53107 (2016).

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