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Extinction of conditioned opiate withdrawal in rats in a two-chambered place conditioning apparatus

Nature Protocols volume 7, pages 517526 (2012) | Download Citation


Conditioned opiate withdrawal contributes to relapse in addicts and can be studied in rats by using the opiate withdrawal–induced conditioned place aversion (OW-CPA) paradigm. Attenuation of conditioned withdrawal through extinction may be beneficial in the treatment of addiction. Here we describe a protocol for studying OW-CPA extinction using a two-chambered place conditioning apparatus. Rats are made dependent on morphine through subcutaneous implantation of morphine pellets and then are trained to acquire OW-CPA through pairings of one chamber with naloxone-precipitated withdrawal and the other chamber with saline. Extinction training consists of re-exposures to both chambers in the absence of precipitated withdrawal. Rats tested after the completion of training show a decline in avoidance of the formerly naloxone-paired chamber with increasing numbers of extinction training sessions. The protocol takes a minimum of 7 d; the exact duration varies with the amount of extinction training, which is determined by the goals of the experiment.

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This work was supported by the National Institute on Drug Abuse (NIDA) (DA012736 to W.A.C. and DA027752 to K.M.M.). The content is solely the responsibility of the authors and does not necessarily represent the official views of NIDA or the US National Institutes of Health.

Author information


  1. Behavioral Genetics Laboratory, McLean Hospital, Belmont, Massachusetts, USA.

    • Karyn M Myers
    • , Anita J Bechtholt-Gompf
    • , Brian R Coleman
    •  & William A Carlezon Jr
  2. Department of Psychiatry, Harvard Medical School, Mailman Research Center, Belmont, Massachusetts, USA.

    • Karyn M Myers
    • , Anita J Bechtholt-Gompf
    • , Brian R Coleman
    •  & William A Carlezon Jr


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K.M.M. developed the concept, designed the experiments, collected data, analyzed the data and wrote the paper; A.J.B.-G. designed the apparatus, gathered pilot data and edited the manuscript; B.R.C. collected data; and W.A.C. developed the concept and edited the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to William A Carlezon Jr.

Supplementary information

Image files

  1. 1.

    Supplementary Fig. 1

    The two-chambered place conditioning apparatus is unbiased. (a) Frequency distributions of time spent on grid and hole floors in naïve rats (n = 141) exposed to the apparatus for the first time were bimodally distributed, such that individual rats tended to prefer one or the other floor type, but as a group the rats showed no systematic bias. (b) Time spent on the grid and hole floors in 12 rats tested seven times. Most rats showed no consistent preference for one or the other floor type. (c) Mean preference scores (defined as time spent on the preferred floor minus time spent on the nonpreferred floor) of the 12 rats whose data are shown in panel b. Error bars indicate SEMs. ANOVA revealed a significant main effect of Test [F(6, 66) = 2.507; p = .030] and a significant linear trend [F(1, 11) = 9.865; p = .009]. All experimental protocols were approved by McLean Hospital's Institutional Animal Care and Use Committee (IACUC).

  2. 2.

    Supplementary Fig. 2

    Assignment of rats to groups. Shown is an example experiment involving four OW-CPA chambers and 12 rats, housed in groups of four, that have been assigned to four experimental groups: extinction plus vehicle or drug, and no extinction plus vehicle or drug. (In the no extinction groups, vehicle or drug is given at the same time as in the extinction groups but the rats are given no further training after OW-CPA acquisition. If the drug is expected to have no effect in nonextinguished animals, the group sizes of the no extinction groups can be halved in anticipation of pooling their data.) Each rat is assigned to a single place conditioing chamber and is run in that chamber for the duration of the experiment. Rats are assigned to groups such that within each group there are an equal number of rats in the grid+ and hole+ conditions (where grid+ indicates that the grid floor is paired with naloxone and hole+ indicates that the hole floor is paired with naloxone). On each of two acquisition days, all rats are exposed first to the saline-paired floor and second to the naloxone-paired floor. On the first extinction day, half of the rats within each extinction group and condition are exposed first to the formerly saline-paired floor and half are exposed first to the formerly naloxone-paired floor. The order of exposure to these floor types is reversed on the second day of extinction training, and reversed again on each subsequent day of extinction training.

  3. 3.

    Supplementary Fig. 3

    Plan for running the acquisition and extinction phases of the example experiment shown in Supplementary Figure 2. For OW-CPA acquisition and extinction training, the chambers are set up with the dividers in place, allowing two rats to be run simultaneously in each chamber. Hence, rats may be injected and placed into the OW-CPA chambers in pairs, as described in the text. Because only one rat from each home cage is assigned to a particular chamber, the pairs of rats are drawn from different home cages, as shown. On each of two acquisition days, all rats are exposed to the saline-paired floor first and the naloxone-paired floor second. On extinction training days the order of exposures is counterbalanced across rats and reversed from one day to the next. Rats receive the same treatment (vehicle or drug) prior to all exposures to each floor during the extinction training phase. Rats in the no extinction groups are injected with vehicle or drug at the same times as rats in the extinction groups, but they are not given any further behavioral training.

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