Name: Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice (Video) | JoVE Journal
Uploaded: 2016-02-18T13:00:00.000+00:00
Duration: 10 min 28 s
Description: Learn conditioned place preference and locomotor behavioral sensitization assays for addiction research using mice. These high-throughput methods enable researchers to study drug reward mechanisms and neurobiological plasticity in vivo.

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5 minutes

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. Automated measurement methods are described; however, it is possible to use non-automated means for each assay (i.e., video recording, hand-scoring).
1. Conditioned Place Preference
<ol>
	<li>Equipment and Room Set-Up:
	<ol>
		<li>Handle experimental mice for 1-3 min&#160;each day, for at least 3-5 days prior to testing. 
		NOTE: Never-handled mice may find removal from the chamber stressful, which can interfere with or alter conditioning.</li>
		<li>Obtain four or more three-chambered CPP apparatuses, preferably equipped with photobeams for automated data collection18. Ensure that each chamber has two larger, visually- and tactilely-distinct chambers connecting to a smaller, neutral chamber via doors that can be raised/lowered to control access. Lids on each chamber should open for insertion/removal of mice and be mounted with small, individually controllable (dimmable) lights (one per chamber). 
		NOTE: CPP chamber designs vary and can be purchased commercially or constructed by researchers. For the &#34;balanced&#34; design, a recommended scheme is one larger chamber with white walls and wire grid flooring and the other with black walls and bar flooring. The middle chamber should have gray walls and solid gray Plexiglass flooring. For explanation purposes, these chambers will be referred to as &#34;white,&#34; &#34;black&#34; and &#34;middle.&#34; Lids should be clear Plexiglass. Alternate compartment configurations (one- or two-chamber) are also possible and discussed elsewhere (see Discussion).</li>
		<li>Setup the room as it will be during testing: turn off or set overhead lights to the dimmest setting and close the door. Use a light meter inside each chamber and set the lid lights so that middle chambers are slightly brighter (15-20 lux) than black and white chambers (6-10 lux) in order to discourage mice from spending time there. 
		NOTE: If the average time spent in the middle is as much or more than in the black or white chambers, further increase the lighting contrast described in Step 1.1.3. Alternatively, use less appealing flooring for the middle. (e.g., ~220 0.5 cm diameter holes, evenly spaced in 6 x 3.5 x &#188;&#34; Plexiglass), but avoid making this chamber aversive. Pilot any alterations to ensure that decreases in middle time are not due to decreases in total explorations (crossings).</li>
		<li>Program automated data collection (&#34;procedure,&#34; Figure 1A) or manually collect data according to the following parameters. Set trials to start upon the first beam break in either conditioning chamber. Set &#34;test&#34; sessions to be 20 min in length and to track time spent and beam breaks in each chamber. Set &#34;conditioning&#34; sessions to be 30 min long and (optionally) to measure beam breaks in each chamber. Set all chamber lights to illuminate during trial.</li>
		<li>Prepare full volume of cocaine solution required for the experiment at hand. Dissolve cocaine HCl in 0.9% NaCl (saline), basing the final concentration on an injection volume of 0.1 ml/10 g body weight (e.g., for a dose of 5 mg/kg, solution concentration is 0.5 mg/ml). Vortex mixture for 30-45 sec, sterile filter (0.2 &#956;m syringe filters) and store at RT.</li>
	</ol>
	</li>
	<li>Testing: 
	NOTE: The general timeline for CPP is PRE-TEST, CONDITIONING, and then POST-TEST. The pre-test may be separated from conditioning by 1-3 days; however, conditioning and the post-test day should take place on consecutive days (Figure 2A). This timing should be kept the same for all cohorts in the same experiment.
	<ol>
		<li>Test during the animals&#39; light phase using the same apparatus for each animal across days.</li>
		<li>Each trial day (i.e., tests and conditioning days), move mice to the behavioral anteroom and allow them to sit undisturbed in their home cages for 1-1.5 hrs before the trial. Choose a location where mice can be moved swiftly from their cage to the apparatus, with minimal disruption, when the trial begins. Turn on all equipment so that any noises associated with the test (e.g., equipment fans) are present.</li>
		<li>Thoroughly clean each apparatus (inner walls, flooring and trays) with mild, alcohol-, glycol-ether- or ammonia-based disinfecting wipes prior to and after each mouse (use the same type of cleaner throughout the experiment). Do not neglect flooring undersides.</li>
		<li>Check that room lights are set appropriately (off or dimmed) at the beginning of each day.</li>
		<li>Proceed with the following according to whether it is a &#34;test&#34; or &#34;conditioning&#34; day:
		<ol>
			<li>On Trial Days 1 and 6 (i.e., the pre- and post-tests), place the inter-chamber doors in the open position (Figure 2B).</li>
			<li>Load the &#34;test&#34; computer program and enter animal IDs (Figure 1A), then issue start command (Figure 1B), if applicable.</li>
			<li>Gently lower each mouse into the middle chamber of its assigned apparatus, facing the back wall, and softly close the lid. Once all chambers are loaded, leave the testing room and minimize noise.</li>
			<li>Leave mice inside each apparatus until trials for all mice have closed/ended (Figure 1C). Export trial data.</li>
		</ol>
		</li>
		<li>Just prior to or following the pre-test, weigh the animals. Use these weights for dose calculations during conditioning.</li>
		<li>In order to prepare for conditioning trials, calculate each mouse&#39;s pre-test &#34;preference&#34; for the black and white chambers by subtracting time spent in each of these from the other (i.e., &#34;black minus white&#34; &#38; &#34;white minus black&#34;; see Figure 3, Columns 9 &#38; 10).</li>
		<li>Since mice with strong initial preferences make balancing difficult, establish an acceptable limit for preference scores (e.g., &#60;33% of total trial time) and exclude mice that exceed it from calculations. Use a liberal limit (&#60;66% of total trial time) to maximize inclusion when attrition is likely after testing is complete (e.g., due to off-target surgical manipulations). 
		NOTE: Mice exceeding the limit can still be tested, with an attempt to keep their pre-test preferences balanced. Later, these mice can be excluded from analysis, if necessary, to balance pre-test scores. If extreme pre-test biases (i.e., &#62;800 sec) disproportionately affect one group, consider altering the conditioning environments and/or using other assays.</li>
		<li>Choose the black or white chamber as the drug-pairing chamber for each mouse, meanwhile summing the corresponding pre-test preference scores within each group with the following priorities in mind: 
		NOTE: Take care to balance scores between groups within each cohort as well as across any previous cohorts.
		<ol>
			<li>Make the sums for all groups as equivalent as possible.</li>
			<li>Make the sums as close to zero as possible (i.e., choose the preferred side for some mice and the non-preferred side for others). If a near-zero sum is impossible for any given group, re-adjust all others to closely match the best obtainable score for the limiting group, favoring slightly negative group sums over positive.</li>
			<li>As much as feasible, keep assignments of black versus white and preferred versus non-preferred chambers for drug pairings even within each group.</li>
			<li>As it is not always possible to meet the above goals, correct any deviations by making opposing balancing considerations in later cohorts; however, try to avoid producing cohorts with wildly different average pre-test preferences.</li>
		</ol>
		</li>
		<li>On Conditioning Days 2-5, place inter-chamber doors in the closed position (Figure 2C).</li>
		<li>Prepare individual syringes with cocaine (Days 2 &#38; 4) or saline (Days 3 &#38; 5) solution, as described in Step 1.1.5 and based on body weights measured at pre-test. 
		NOTE: Dose should be chosen with regard to experimental expectations, considerations mentioned in the Introduction, and potential floor and ceiling effects. It is often best to conduct independent experiments using at least two different doses.</li>
		<li>Load &#34;conditioning&#34; computer program and enter animal IDs (Figure 1A), then issue start command (Figure 1B), if applicable.</li>
		<li>Scruff and inject each mouse (i.p.), immediately lowering them into the appropriate black or white chamber of their assigned apparatus facing the back wall, then softly close the lid. Once all chambers are loaded, leave the testing room and minimize noise.</li>
		<li>Remove mice from their chamber as close to exactly 30 min as feasible (i.e., the first mice are removed from their chambers while other mice are still conditioning). Remove animals as quietly as possible, without introducing noise.</li>
	</ol>
	</li>
	<li>Statistical Analysis:
	<ol>
		<li>Choose a method of analysis. Either subtract time spent on the saline-paired side during the post-test from time spent on the cocaine-paired side during the post-test (cocaine - saline, sec) or use the time spent in the post-test drug-paired chamber minus the pre-test time spent on the drug-paired chamber. 
		NOTE: if using the first method, also plot line graphs of average time spent in the middle, saline- and drug-paired chambers during the pre- and post-tests for each group. Compared to the pre-test, the post-test should show increased time in the drug-paired side and decreased time spent in the saline-paired side (see Figure 6, bottom, and Discussion for explanation).</li>
		<li>Depending upon the nature and number of groups being compared, use a t-test, One- or Two-Way ANOVA, as appropriate, possibly with post-hoc analysis, to analyze either of the above-presented subtraction scores. 
		NOTE: Cocaine preference scores tend to be variable, and furthermore, can be negative (i.e., indicate aversion to the drug-paired side). Mice that show aversion should not be removed (unless they are statistical outliers), since this result is normal and likely important to determining differences between groups. Expect to need sample sizes of 12 to 30 animals per group, depending on the effect size of the treatment.</li>
	</ol>
	</li>
</ol>
2. Locomotor Sensitization
<ol>
	<li>Equipment and Room Set-Up
	<ol>
		<li>Obtain a 4 x 8 (X x Y) photobeam array (outside dimensions 11.5&#34; x 20&#34;). Construct an open-topped chamber made of black Plexiglas (inner dimensions 22 1/6&#34; x 13 &#190;&#34; x 9 3/8&#34;) to house the array (Figure 4A).</li>
		<li>Prepare the testing room so that a red light (ceiling- or wall-mounted) can be used during testing.</li>
		<li>Prepare separate program sessions for daily habituation and injection trials.
		<ol>
			<li>Set habituation trials to be between 30-60 min and injection trials to be between 60-120 min (Figure 5B). The recommended length for each is 60 min. Keep trial length consistent across multiple cohorts within the same experiment.</li>
			<li>Set trials to start recording upon the first beam break that occurs after the start signal has been initiated (Figure 5B). For the injection trial, set the start signal so that boxes can be started individually (not in unison), if possible.</li>
			<li>Set beam breaks to be recorded in user-defined &#34;bins,&#34; preferably 5 min each (Figure 5B).</li>
		</ol>
		</li>
		<li>Prepare full volume of cocaine solution required for the experiment at hand. Dissolve cocaine HCl in 0.9% NaCl (saline), basing the final concentration on an injection volume of 0.1 ml/10 g body weight (e.g., for a dose of 5 mg/kg, solution concentration is 0.5 mg/ml). Vortex mixture for 30-45 sec, sterile filter (0.2 &#956;m syringe filters) and store at RT.</li>
	</ol>
	</li>
	<li>Testing 
	NOTE: The initial phase of testing runs for 10-11 consecutive days. Mice receive two daily trials: habituation (injection-free) and injection. Administer saline for the first three to four days (see Discussion for importance of saline habituation), and cocaine for the next seven using the same dose (e.g., 15 mg/kg/day).
	<ol>
		<li>Each day, acclimate mice in their home cages to the behavioral anteroom for 30 min to 1 hr.</li>
		<li>Prepare clean standard mouse-sized clear acrylic housing cages with an extremely thin layer of fresh bedding (e.g., pine chips), so as to not obscure photobeams, if applicable (Figure 4C &#38; D).</li>
		<li>Place cages against the Y-axis of the photobeam array near one end, so that five beams are spaced evenly along its length. X-axis beams are not used in this test (Figure 4C &#38; D).</li>
		<li>Take mice from their home cage, in random order, scruff and weigh them. Remove each mouse from the weigh boat by the base of their tail (with support) and place directly into their assigned locomotor cage. Cover each cage with a standard filter-top lid.</li>
		<li>Prepare individual syringes with saline or cocaine solution, as described in Step 2.1.7, based on the current day&#39;s body weights. Begin with a dose of 15 or 20 mg/kg, and consider a second experiment using a higher or lower dose (see Discussion for relevant factors).</li>
		<li>Once habituation trials have ended for all cages, load the injection program.</li>
		<li>One at a time, remove mice from their test cage, scruff and give their injection (i.p.). Before returning the mouse to its test cage, quickly initiate the start signal for that cage (Figure 5C, bottom).</li>
		<li>After all injection trials have ended, return mice to their home cages and their housing room.</li>
		<li>If desired, allow mice to undergo a series of withdrawal periods and drug challenges, such as the following: same dose as original, half-dose, double-dose, then saline, allowing seven days before the same-dose challenge and three to seven days before each additional challenge. Whatever the choice of challenges, maintain similar withdrawal periods across cohorts within an experiment. 
		NOTE: If the original dose is high (e.g., 30 mg/kg or above), the double-dose should be skipped or replaced with a lower dose. The saline challenge reveals any conditioned locomotor activation by the cocaine-paired environment alone, and in the process, the amount of sensitized locomotion that is cocaine-dependent (see Discussion).</li>
	</ol>
	</li>
	<li>Statistical Analysis
	<ol>
		<li>Choose the portion(s) of each trial that will be used for analysis. Most cocaine-induced locomotion in rodents occurs within the first ~15-30 min after drug injection. Depending on involved variables, consider analyzing cumulative locomotion for multiple time windows (e.g., the first 15, 30, 60 and/or 120 min) or focus on independent segments of the trial.</li>
		<li>Using the time frame chosen, sum the beam breaks for each animal by day for habituation and injection trials separately and then average the sums for each group. Plot means and standard errors of the mean (S.E.M.) in a line graph over all days. As treatments may alter how mice respond to the drug early and/or late in each daily trial, also plot average group beam breaks by 5-min bin over the course of each daily trial. 
		NOTE: Plotting daily activity averages for habituation and injection trials (e.g., first 15 min) makes it artificially appear that locomotion is dampened by saline injection, but remember that activity has (most likely) declined to this level by the end of habituation each day.</li>
		<li>Analyze consecutive saline and drug treatment days separately using repeated measures (RM) ANOVAs having the within-subjects factor of day/time and including any between-subjects factors in the design, as appropriate. Use a t-test or One-Way ANOVA to investigate group differences on Day 1 of cocaine (acute exposure), and a Multivariate ANOVA for the challenges. When appropriate, follow significance with post-hoc tests or Univariate ANOVAs.</li>
	</ol>
	</li>
</ol>

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<li>Li, Y., Acerbo, M. J., Robinson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+induction+of+behavioural+sensitization+is+associated+with+cocaine-induced+structural+plasticity+in+the+core+%28but+not+shell%29+of+the+nucleus+accumbens%5BTitle%5D)+AND+%22Eur+J+Neurosci%22%5BJournal%5D)">The induction of behavioural sensitization is associated with cocaine-induced structural plasticity in the core (but not shell) of the nucleus accumbens.</a> Eur J Neurosci. 20 (6), 1647-1654 (2004).</li>
<li>Partridge, B., Schenk, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Context-independent+sensitization+to+the+locomotor-activating+effects+of+cocaine%5BTitle%5D)+AND+%22Pharmacol+Biochem+Behav%22%5BJournal%5D)">Context-independent sensitization to the locomotor-activating effects of cocaine.</a> Pharmacol Biochem Behav. 63 (4), 543-548 (1999).</li>
<li>Le Foll, B., Diaz, J., Sokoloff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+dopamine+D3+receptor+expression+accompanying+behavioral+sensitization+to+nicotine+in+rats%5BTitle%5D)+AND+%22Synapse%22%5BJournal%5D)">Increased dopamine D3 receptor expression accompanying behavioral sensitization to nicotine in rats.</a> Synapse. 47 (3), 176-183 (2003).</li>
<li>Heidbreder, C. A., Babovic-Vuksanovic, D., Shoaib, M., Shippenberg, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+behavioral+sensitization+to+cocaine%3A+influence+of+kappa+opioid+receptor+agonists%5BTitle%5D)+AND+%22J+Pharmacol+Exp+Ther%22%5BJournal%5D)">Development of behavioral sensitization to cocaine: influence of kappa opioid receptor agonists.</a> J Pharmacol Exp Ther. 275 (1), 150-163 (1995).</li>
<li>Tirelli, E., Michel, A., Brabant, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cocaine-conditioned+activity+persists+for+a+longer+time+than+cocaine-sensitized+activity+in+mice%3A+implications+for+the+theories+using+Pavlovian+excitatory+conditioning+to+explain+the+context-specificity+of+sensitization%5BTitle%5D)+AND+%22Behav+Brain+Res%22%5BJournal%5D)">Cocaine-conditioned activity persists for a longer time than cocaine-sensitized activity in mice: implications for the theories using Pavlovian excitatory conditioning to explain the context-specificity of sensitization.</a> Behav Brain Res. 165 (1), 18-25 (2005).</li>
<li>Anagnostaras, S. G., Schallert, T., Robinson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Memory+processes+governing+amphetamine-induced+psychomotor+sensitization%5BTitle%5D)+AND+%22Neuropsychopharmacology%22%5BJournal%5D)">Memory processes governing amphetamine-induced psychomotor sensitization.</a> Neuropsychopharmacology. 26 (6), 703-715 (2002).</li>
<li>Spangler, R., Zhou, Y., Schlussman, S. D., Ho, A., Kreek, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+stereotypies+induced+by+%27binge%27+cocaine+administration+are+independent+of+drug-induced+increases+in+corticosterone+levels%5BTitle%5D)+AND+%22Behav+Brain+Res%22%5BJournal%5D)">Behavioral stereotypies induced by 'binge' cocaine administration are independent of drug-induced increases in corticosterone levels.</a> Behav Brain Res. 86 (2), 201-204 (1997).</li>
<li>Kelley, A. E. Measurement of rodent stereotyped behavior. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKelley%2C+AE+%22Curr+Protoc+Neurosci%22">Curr Protoc Neurosci</a>. Chapter 8, Unit 8.8(2001).</li>
<li>Taniguchi, M., Carreira, M. B., Smith, L. N., Zirlin, B. C., Neve, R. L., Cowan, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+deacetylase+5+limits+cocaine+reward+through+cAMP-induced+nuclear+import%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Histone deacetylase 5 limits cocaine reward through cAMP-induced nuclear import.</a> Neuron. 73 (1), 108-120 (2012).</li>
<li>Zangen, A., Solinas, M., Ikemoto, S., Goldberg, S. R., Wise, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+brain+sites+for+cannabinoid+reward%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Two brain sites for cannabinoid reward.</a> J Neurosci. 26 (18), 4901-4907 (2006).</li>
<li>Huston, J. P., de Souza Silva, M. A., Topic, B., Müller, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((What%27s+conditioned+in+conditioned+place+preference%5BTitle%5D)+AND+%22Trends+Pharmacol+Sci%22%5BJournal%5D)">What's conditioned in conditioned place preference.</a> Trends Pharmacol Sci. 34 (3), 162-166 (2013).</li>
<li>Schechter, M. D., Calcagnetti, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trends+in+place+preference+conditioning+with+a+cross-indexed+bibliography%3B+1957-1991%5BTitle%5D)+AND+%22Neurosci+Biobehav+Rev%22%5BJournal%5D)">Trends in place preference conditioning with a cross-indexed bibliography; 1957-1991.</a> Neurosci Biobehav Rev. 17, 21-41 (1993).</li>
<li>Bevins, R. A., Cunningham, C. L. Chapter 9, Place Conditioning: A Methodological Analysis. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBevins%2C+RA+%22Tasks+and+Techiniques%3A+A+sampling+of+methodologies+for+the+investigation+of+animal+learning%2C+behavior%2C+and+cognition%22">Tasks and Techiniques: A sampling of methodologies for the investigation of animal learning, behavior, and cognition</a>. Anderson, M. , 99-110 (2006).</li>
<li>Hitchcock, L. N., Cunningham, C. L., Lattal, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cue+configuration+effects+in+the+acquisition+of+a+cocaine-induced+place+preference%5BTitle%5D)+AND+%22Behav+Neurosci%22%5BJournal%5D)">Cue configuration effects in the acquisition of a cocaine-induced place preference.</a> Behav Neurosci. 128 (2), 217-227 (2014).</li>
<li>Liu, Z. -H., Chuang, D. M., Smith, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lithium+amerliorates+phenotypic+deficits+in+a+mouse+model+of+fragile+X+syndrome%5BTitle%5D)+AND+%22Int+J+Neuropscyhopharmacol%22%5BJournal%5D)">Lithium amerliorates phenotypic deficits in a mouse model of fragile X syndrome.</a> Int J Neuropscyhopharmacol. 14 (5), 618-630 (2011).</li>
<li>Bardo, M. T., Rowlett, J. K., Harris, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conditioned+place+preference+using+opiate+and+stimulant+drugs%3A+A+meta-analysis%5BTitle%5D)+AND+%22Neurosci+Biobehav+Rev%22%5BJournal%5D)">Conditioned place preference using opiate and stimulant drugs: A meta-analysis.</a> Neurosci Biobehav Rev. 19 (1), 39-51 (1995).</li>
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The authors declare that they have no competing financial interests.

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

assessment of cocaine-induced behavioral sensitization and conditioned place preference in mice

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&lt;0.05), which was interpreted in lieu of significant main effects observed for both Test...

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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Research

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Behavior

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans.  In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

Novel Apparatus and Method for Drug Reinforcement

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse.  It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

Operant Sensation Seeking in the Mouse

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Methods for Intravenous Self Administration in a Mouse Model

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.

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

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.

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

Behavioral Assessment of the Aging Mouse Vestibular System

Learn conditioned place preference and locomotor behavioral sensitization assays for addiction research using mice. These high-throughput methods enable researchers to study drug reward mechanisms and neurobiological plasticity in vivo.

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

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