Conditioned Locomotion Is Not Correlated with Behavioral Sensitization to Cocaine: An Intra-Laboratory Multi-Sample Analysis Gregory Hotsenpiller, Ph.D., and Marina E. Wolf, Ph.D. Pre-clinical and clinical studies have demonstrated the of training, all subjects were tested with the conditioned importance of associative factors in regulating craving for stimuli in the absence of drug and conditioned locomotion drugs of abuse. To model these conditioned effects, we have was measured. The response of Unpaired subjects on the last examine cue-induced conditioned locomotion in rodents. The training day was positively correlated with their response on present study involved analysis of several of our prior studies test day, as expected since both days were nearly identical to evaluate the relationship between conditioned locomotion (stimuli presented without cocaine). However, for Paired and behavioral sensitization using a within-subjects analysis. subjects, the magnitude of conditioned locomotion on the Both are animal models used to study addiction, so it is drug-free test day was not positively correlated with the important to know if one is predictive of the other, and more magnitude of behavioral sensitization. These results generally, if drug effects are predictive of conditioned effects. underscore the importance of focusing research on drug-free In all of our studies, Paired subjects received cocaine during conditioned behaviors when attempting to model conditioned presentation of conditioned stimuli while Unpaired subjects responses to drug related cues in human addicts. received saline with the stimuli and cocaine at the home cages [Neuropsychopharmacology 27:924–929, 2002] an hour later. Paired subjects typically displayed behavioral © 2002 American College of Neuropsychopharmacology. sensitization over the course of training. After the completion Published by Elsevier Science Inc. KEY WORDS: Addiction; Associative; Conditioning; have demonstrated that humans trained in a laboratory Craving; Pavlovian; Sensitization setting to associate cocaine with novel non-drug related stimuli will exhibit conditioned craving upon presenta- Addiction to drugs of abuse resembles the pattern of a tion of the conditioned stimuli (CSϩ). chronic illness (McLellan et al. 2000). Much of the prob- Because of the importance of conditioning in drug use, lem in treating drug addiction is craving, often brought our laboratory has developed a rodent model of condi- on by conditioned cues, which can lead to relapse (e.g. tioned locomotion. Paired subjects receive cocaine with the O’Brien et al. 1998). Indeed, Foltin and Haney (2000) conditioned stimuli while Unpaired subjects receive saline with the stimuli and cocaine at the home cages an hour From the Department of Neuroscience, FUHS/The Chicago Med- later. On test day, we measure a drug free conditioned re- ical School, 3333 Green Bay Road, North Chicago, IL 60064-3095. sponse to stimuli formerly paired with cocaine. The utility Address correspondence to: Dr. Marina E. Wolf, Department of of this model is that the conditioned behavior is not goal Neuroscience, FUHS/The Chicago Medical School, 3333 Green Bay Road, North Chicago, IL, 60064-3095. Tel.: (847) 578-8659; Fax: (847) directed, as in operant behavioral studies (Olmstead et al. 578-8515; E-mail: [email protected] 2001), and therefore is not complicated by response-re- Received February 27, 2002; revised April 25, 2002; accepted May ward expectancies. We have used this model to explore the 2, 2002. Online publication: 5/9/02 at www.acnp.org/citations/ neurochemical basis of drug conditioned responses (Hot- Npp050902304. senpiller et al. 2001, 2002; Hotsenpiller and Wolf 2002). NEUROPSYCHOPHARMACOLOGY 2002–VOL. 27, NO. 6 © 2002 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 0893-133X/02/$–see front matter 360 Park Avenue South, New York, NY 10010-1710 PII S0893-133X(02)00370-6 NEUROPSYCHOPHARMACOLOGY 2002–VOL. 27, NO. 6 Behavioral Sensitization and Conditioned Locomotion 925 Of course, Pavlovian models, such as ours, do not ad- details). All procedures were conducted in accordance dress whether the performance of the conditioned re- with the National Institutes of Health Guide for the sponse is associated with desire to obtain the drug. How- Care and Use of Laboratory Animals. All subjects were ever, this seems likely based on results of place-preference given a total of six cocaine injections, which were either studies (e.g. Bardo and Bevins 2000) and studies of cue-in- contingent with the presentation of the stimuli (Paired, duced reinstatement of cocaine seeking behavior (Grimm n ϭ 44) or not (Unpaired, n ϭ 44). In some studies (dis- and See 2000). Furthermore, several studies have demon- crete cue conditioning), subjects received intervening strated the ability of classically conditioned stimuli to sub- no-stimuli days during training. See the studies cited sequently control drug-seeking behavior following extinc- in Table 1 for details of each conditioning regimen. The tion of the operant response (Alleweireldt et al. 2001; present analysis was based on 30-min cumulative am- Kruzich et al. 2001; Crombag and Shaham 2002). These ex- bulation counts measured during the first presentation amples indicate that classically conditioned (Pavlovian) of stimuli (Day 1 CSϩ), the last presentation of stimuli cues can elicit drug-seeking behavior in animals. (Day 6 CSϩ), and test sessions for conditioned locomo- During the course of training, Paired subjects are ex- tion in the presence of the stimuli (conducted 3 to 10 posed repeatedly to cocaine and typically develop behav- days after the last presentation of stimuli). The latter ioral sensitization to its locomotor activating effects. Given test sessions provided a measure of the magnitude of that both conditioned locomotion and sensitization are ani- conditioned locomotion for each rat. Two related but mal models used to study the neurobiology of drug abuse, distinct measures were used to obtain an index of sensi- it would be valuable to determine the relationship between tization. The ratio of ambulations on Day 6 CSϩ to these two phenomena. More generally, it is important to those on Day 1 CSϩ (multiplied by 100) was used as a determine whether responses to drugs themselves (e.g. measure of the magnitude of sensitization that devel- sensitization) are predictive of responses to drug condi- oped during training. The number of ambulations on tioned cues. In our initial study, which characterized a Day 6 CSϩ was used as a measure of the magnitude of model in which cocaine was paired with discrete cues, we the sensitized response to cocaine challenge. performed correlation analyses to assess whether the mag- Locomotor activity was measured using photobeam nitude of conditioned locomotion was related to other be- frames (30x50 cm) situated around Plexiglas cages and havioral measures during habituation sessions, training interfaced with an analysis system (PAS, San Diego In- sessions (i.e. cocaine responses for paired subjects), and co- struments, San Diego, CA). Three pairs of photobeams caine extinction/challenge tests (Hotsenpiller et al. 2001). were evenly spaced on the long axis of the frame. When Although none of the variables were significantly corre- two adjacent beams were broken in succession, this was lated with conditioned locomotion, a subtle relationship counted as an ambulation. may have been missed given the relatively small sample size (n ϭ 11 rats/group). Therefore, the present multi-sam- Data Analysis ple analysis of our conditioned locomotion research was conducted to further examine the relationship between To test for any change in behavioral response over the sensitized behaviors and conditioned locomotion. No course of the training sessions, independent groups t- study to date has performed an analysis of this magnitude tests were conducted comparing the ambulations of on the relationship between unconditioned and condi- Paired or Unpaired subjects on Day 1 CSϩ to those on tioned effects of cocaine. Furthermore, aside from our ini- Day 6 CSϩ. t-tests were conducted to compare ambula- tial analysis (Hotsenpiller et al. 2001), no study has used a tion counts of Paired and Unpaired subjects during within-subjects analysis to investigate the relationship be- tests of conditioned locomotion. To determine if the de- tween unconditioned drug effects and conditioned loco- gree of conditioned locomotion was related to behav- motion. The importance of such an analysis is in the power ioral sensitization, Pearson correlation coefficients were derived from large sample sizes and within-subjects data, used to compare subjects’ ambulations during the test which increases the likelihood of uncovering significant as- for conditioned locomotion with: (1) the ratio of ambu- sociations. Furthermore, the fact that the unconditioned lations on Day 6 CSϩ to those on Day 1 CSϩ; and (2) and conditioned responses measured are the same (ambu- the number of ambulations on Day 6 CSϩ. lations) could facilitate detection of a relationship. RESULTS MATERIALS AND METHODS Training and Testing for Conditioned Locomotion Subjects and Design Figure 1 (left panel) shows the ambulations for the Subjects were male Sprague-Dawley rats (Harlan Spra- Paired and Unpaired subjects during the first (Day 1 gue Dawley, Indianapolis, IN, colony 205) used in our CSϩ) and last (Day 6 CSϩ) days of training. As ex- prior studies of conditioned locomotion (see Table 1 for pected,