Conditioned Place Aversion Protocol

General Overview
Conditioned place aversion (environmental place aversion) is commonly used to evaluate aversions for environmental stimuli that have been associated with a negative reward. In general, this procedure involves several trials where the animal is presented with a negative stimulus (e.g., illness or withdrawal from drug dependence), paired with placement into a distinct environment containing various cues (e.g., tactile, visual, and/or olfactory). When later tested in the normal state, withdrawals and the amount of time spent in the compartments previously associated with the negative stimulus serves as an indicator of preference and a measure of reward learning.

Apparatus
Each place conditioning apparatus consists of an open field enclosed in separate light- and sound-attenuating chambers. General activity and location in the open field is monitored by video recording. The floor of the open field consists of interchangeable halves made of one of two textures. The combination of floor textures was selected on the basis of previous studies showing that mice spend an average of about 50% time on each floor type during preference tests (Cunningham et al. 1992, 1997; Cunningham 1995). Thus, the apparatus is "unbiased". Specifically, the floors are either a "grid" made from stainless-steel rods or a "hole" floor made from perforated stainless steel. Prior to each session the open field and floors are cleaned using a 70% ETOH solution.

Procedure
Each place conditioning experiment consists of the following four phases.

(1) Habituation phase (2 days):
On two 30-min sessions (1 session/day) mice are able to freely explore the entire open field apparatus using a non-discriminative flat plastic floor.

(2) Pre-conditioning bias test (1 day):
To determine whether a pre-existing bias exists, a single 30 min test session is conducted during which mice are allowed to freely explore the open field with half of the arena containing the "grid" floor and half of the arena containing the "hole" floor.

(3) Conditioning (max 8 days):
Mice are randomly assigned to one of two groups For one group, placement into the apparatus with one floor-type is paired with a negative stimulus and for a second group the apparatus floor is paired with a placebo procedure. All mice receive four 30-60 min conditioning sessions of this type, one session every second day. On the intervening days, mice are exposed to the alternate floor type and given a placebo treatment. On all sessions, mice have access to the entire apparatus with the same floor texture on both sides.

(4) Post-conditioning bias test (3 days):
The 30-min floor aversion tests are conducted 24h, 7d and 30d after the last conditioning session. The conditions of these tests are identical to those during the pre-conditioning bias test. The number of seconds spent on the target (previously paired) floor is measured and the results are compared with those from the pre-conditioning bias test.

All aversion conditioning experiments follow this procedure but differ in the reinforcing stimulus used during the conditioning phase. Examples of experiments using either an illness-induced negative reinforcing stimulus or a withdrawal-induced negative reinforcing stimulus are described below:

1. Illness place aversion conditioning
During the conditioning phase, mice are randomly assigned to a Lithium Chloride (LiCl)-paired or a control group. LiCl-paired mice receive an IP injection of LiCl, and control mice receive an IP injection of saline, prior to placement on one of the floor types. All mice receive four 30-min conditioning sessions of this type, one session every other day. On intervening days, all mice receive an injection of saline prior to placement on the alternate floor type.

2. Morphine-withdrawal place aversion conditioning
Prior to the start of the experiment half of the mice are implanted with osmotic pumps containing morphine and half undergo sham surgery. During the conditioning phase, for each group mice are randomly assigned to a naltrexone-paired or a saline-control group. Because prior studies indicate that a single experience with opiate withdrawal is sufficient to produce the development of robust conditioned place aversion, mice undergo only two sessions - one session during which the Naltrexone group receive a single injection Naltrexone and the control group receive saline, paired with one floor type; and a second session when all mice receive saline paired with the alternate floor type. Thus, each mouse undergoes 1 experience of precipitated withdrawal. Both Naltrexone and saline are injected SC. Mice remain in the open field for 30 min each session.