CPP is a form of Pavlovian conditioning that is used to investigate the rewarding effects associated with drugs of abuse.
Adult zebrafish are conditioned to associate drug exposure with specific (neutral) environmental stimuli. The aim of this experiment is to measure the amount of time spent in a particular environment that has previously been associated with a substance (drug), indicating positive-reinforcing qualities of that compound.
The Zantiks AD unit is used for CPP in zebrafish. The dividing inserts are used to create four separate testing compartments for tracking four fish in one unit (see image A above). Stimuli are presented from the integrated screen below the testing tank. The stimuli can be comprised of colour, shapes, or stripes and be both biased and unbiased in presentation (see image B).
The CPP paradigm is conducted in three testing phases occurring on consecutive days.
- *IMPORTANT Single housing: In order to reduce any stress effects, the fish to be tested should be singly housed for one week prior to a habituation phase.
- Habituation/Autoshaping: For two days before recording CPP, individual fish should also be gently netted and placed in the Zantiks AD tank and allowed to swim freely for 10-20 minutes. This step allows the fish to habituate to the new testing environment.
- Baseline: In the first testing phase, baseline preferences for two distinct environments are recorded. Fish are placed into individual arenas for a 20 minute period, during which, each fish is allowed to explore its full arena with both stimuli shown together. The Zantiks AD system can automatically track and measure the time spent and distances travelled within the two stimuli environments to calculate a baseline preference.
- Calculation of the percentage and baseline stimulus preference. The proportion of time spent in the vicinity of each of the stimuli for baseline is calculated as: Time in Stimulus A / (Time in Stimulus A + Time in Stimulus B).
- Conditioning: Following establishment of baseline preferences, on three consecutive days, the fish are exposed to the test compound or control treatment in the presence of the least-preferred stimulus (stimulus in which the fish spent the least amount of time during baseline). Fish are placed into arenas individually, with the preferred stimulus (stimulus in which the fish spent the most time during baseline) projected for 20 minutes. The stimulus is then changed to the least-preferred stimulus for 20 minutes and the test compound or saline is administered into the tank.
- Fish are returned to home tank, and testing tank is washed out.
- Probe: After the 3 days of conditioning, any change in preference is determined using a probe trial. This step is identical to the baseline recordings. Fish are placed into the Zantiks AD tank for a 20 minute period. Both stimuli are presented together, and the fish has free range of the arena. The amount of time and distance travelled within either stimulus environment is measured.
- Calculation of the percentage and probe stimulus preference. Change in place preference (CPP Change Score) is calculated by subtracting the percentage of time spent in the drug-paired environment before conditioning from the percentage of time in the drug-paired environment after conditioning, and expressed as a percentage.
Results / Data output
The behavioural endpoints typically measured in this task are time spent in the vicinity of each stimuli and distance travelled. The Zantiks AD system can automatically measure and process these behavioural endpoints in an easy to read format.
Processed data files are written as .csv files allowing access from any spreadsheet program, such as Excel, Google docs or Numbers. The typical variables used in CPP protocols include distance travelled, time spent in each variable , counters , and total distance travelled.
An increase in time spent in the drug-paired environment relative to a control is taken as evidence that the test compound was rewarding (calculated as the CPP Change Score). The learned association between the environmental stimulus and the test compound results in the fish spending more time in that environmental stimulus.
CPP change scores are frequently analysed by a one-way ANOVA comparing doses of treatment conditions (see images D & E).