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.

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			<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>
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			<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>
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			<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>
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			<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>
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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 ...

The overall goal of these behavioral tasks is to allow researchers to assay drug-related behaviors such as reward and sensitization.These methods can help answer key questions in the drug addiction field, such as which molecules or pathways contribute to drug-induced plasticity.The main advantage of these techniques is that they are high throughput and can be conducted using either home made or purchased equipment.When we do perform conditioned place preference assays using different drug doses, this approach can uncover differences resulting from altered drug sensitivity and provide additional confidence in your findings.This experiment makes use of three chambered CPP boxes equipped with photo beams for automated data collection.Have four or more on hand.This apparatus has two large chambers, A and B, into which mice can be introduced through a lid.Within each chamber is a dimmable light source.The A and B chambers are made visually distinct, for example, white versus black, and also tactilely distinct, such as with different flooring textures.Between the two large chambers is a smaller, interconnecting neutral chamber, or C, which is accessible by sliding doors.For further details on the CPP boxes, including important information on how to set up a balanced design, consult the text protocol.For these experiments, use mice that have undergone regular handling to minimize the impact of variable animal handling on the results.Prepare the test room, clean the CPP boxes thoroughly.Turn on the equipment and dim the room lights.Then, move the mice to the test area 60 to 90 minutes before the trial.On the first testing day, perform a pre-test.Prepare the chambers by opening the inter-chamber doors.Next, open the computer program.Load the test program.Therein, enter the animal IDs and issue the start command.When ready, gently place each mouse into the middle chamber of their assigned apparatus.After the pre-trials, return the mice to their home cages and export the data.From the pre-test results, determine each animal's preference for chambers A and B.Subtract the time spent in each from the other, and use the difference as an index.Give special consideration to animals with strong pre-test preferences, shown here in red, as described in the text protocol.Select the drug paired chamber for each mouse based on this result.The sum of the index scores for each test group should be made equivalent and near zero.In other words, choose the preferred side for about half of the mice.Also, assign animals evenly to the A and B chambers within each group.If conditioning multiple groups simultaneously is desirable, attempt to assign animals that are using the same apparatus to opposite chambers.For more details, consult the text protocol.A critical step in conditioned place preference when using a balanced design is assignment of the drug paired chambers so that pre-test scores are nearly equal for all test groups and as close to zero or no preference as possible.To begin, always weigh each mouse before the conditioning phase.On the first conditioning day, prepare syringes with cocaine or the chosen test drug according to each animal's body weight.On alternate days, prepare syringes with saline or another appropriate vehicle.Prepare the boxes by closing the inter-chamber doors.Load the conditioning program on the computer, enter the IDs, and start the program.Next, inject the animals with their drug or vehicle bolus and load them into their assigned box.It is critical to ensure that conditioning occurs in the proper A or B chambers for each mouse, each day, as assigned, since errors in this step can force removal of animals from the study.After no more than 30 minutes of conditioning, return the mice to their home cages.When all the trials are complete, export the data and return the mice to the colony.This assay makes use of four by eight photo beam arrays that are 20 inches by 11.5 inches.Have between four and 16 on hand.House each array in an open top black Plexiglas chamber.In the testing room, have a red light prepared to illuminate the chambers.In the software, prepare the program sessions for daily habituation and injection trials.First, set the habituation trial length to be 30 to 60 minutes.An hour is a good starting point.Have the beam breaks recorded in five minute or shorter bins.Second, set the time of the trial in each chamber to begin when the first photo beam is tripped, which comes after the trial has been initiated with a start signal.Third, set the injection trial length to be 60 to 120 minutes.If possible, for injection trials, choose to initiate the start signals from the photo beam arrays individually and not in unison.Locomotor sensitization requires an initial testing run of 10 or 11 consecutive days.On each day, bring the mice into the behavioral anteroom 30 to 60 minutes before the test.Now, place a fresh translucent mouse cage into each chamber.Make sure that the photo beams are centered on the long axis of each cage.When ready, remove the mice in random order from their home cages.First, scruff and weigh them.Then place each into their assigned locomotor testing cage for the habituation trial and cover with a lid.Each day involves a habituation trial with no injection, followed by an injection trial.During the habituation trial, prepare syringes based on each mouse's current body weight.After all habituation trials have ended, load the injection trial program and quietly enter the testing room.After injecting a mouse, quickly issue the cage start signal in the software, and then return that mouse to its test cage.Over the first three to four test days, administer saline or another appropriate vehicle.Over the next seven days, administer cocaine or the chosen test drug.After all the injection trials have ended, return the mice to their housing room.In the CPP assay, wild type C57 black 6n mice were conditioned with saline only, or two different concentrations of cocaine.The saline only group showed no change in preference after conditioning, as expected, while enhanced preferences were observed in groups treated with cocaine.The time spent in different chambers pre-and post-test demonstrated that more time was spent in the cocaine-paired chamber by the cocaine treated mice than in either other chamber.Mice that received only saline showed no striking differences in chamber preference, as expected.Next, the locomotor assay was performed on C67 black 6n mice.Saline injections were given for three to four days, followed by cocaine injections at four different doses for seven days.As expected, more pronounced changes in behavior were observed with increasing doses of cocaine.For a challenge, after one to two weeks of withdrawal, each group was given the original dose of cocaine.Additional challenges were then given with variable withdrawal periods and doses.Although this video demonstrated these techniques using commercially purchased equipment, many labs successfully performed them using home-constructed devices.While attempting these procedures, it is important to ensure that all animals receive equal treatment in order to reduce stress, errors and inter-experiment variability.Variables to consider include handling time and time spent in the testing room and chamber.Extensions of these assays including extinction and reinstatement for CPP and long term or cross-sensitization challenges for locomotor sensitization may be desirable.In addition, its important to use drug na

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