Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

*Most important steps.

1. Equipment and Software

1. Training and testing is carried out in mouse operant chambers equipped with two ultra-light, retractable levers¹ positioned on either side of a food receptacle (see Table 1). A stimulus light is located above each lever and there is a house light at the top of the cage. A pellet dispenser is located outside of the chamber. Each chamber is housed inside a sound-attenuating cubicle with ventilating fan. Chambers are connected to a PC computer with MedPC IV software via a “Smart Control” interface cabinet.

Note: When ultra-light levers are not available it is recommended that nose-poke be used as the operant response for mouse studies. However, with both options available we suggest selection of ultra-light levers as a more reliable measure of motivation given that nose-poke responses can be triggered by exploratory sniffing.

2. The MedPC programs used to run operant tasks are written in Medstate notation code which a user can learn with the help of the programming manual provided. Programming provides flexibility for the user to modify existing codes or generate unique experimental parameters. Certain codes can be obtained for free from Med Associates “Medstate Notation Repository” http://www.mednr.com. Additional codes may be purchased from Med Associates.

2. Mouse Acclimation and Food Restriction

1. Upon arrival mice are group-housed for at least 10 days to acclimatize to a reverse 12 h light/dark cycle and provided with ad libitum access to standard chow and water. In our case we use adult C57BL/6 mice purchased from Charles River (St. Constant, QC). All mouse manipulations and behavioral experiments are performed during the dark cycle. All procedures involving the use of animals were approved by the CRCHUM Animal Care Committee.
2. Mice are subsequently singly-housed and food restricted (70% of their daily food intake) until they reach 90-95% free-feeding body weights in order to facilitate acquisition of lever-press responding. Mice were provided their daily quota of food in the home cage after the termination of session. Upon reaching 5-10% weight loss, the daily food allotted is adjusted to stabilize the lower body weights for the remainder of the training period. Once mice have attained acquisition criteria (see below) they are put back on an ad libitum feeding schedule. Mice regain lost body weight in about 3-4 days.
3. Mice are handled in the test room for 3 consecutive days prior to the first training session. The day prior to the start of training 10-15 high-fat and high-sugar (HFHS) pellets are placed into the home cage to prevent the potential influence of food neophobia on operant performance. We use 20 mg HFHS, dust-free, precision food pellets (14 mg also available) containing 48.9% Kcal as fat (Bio Serv, Frenchtown, NJ).

3. Operant Training

1. Mice are initially trained to press the lever on a fixed ratio (FR)-1 reinforcement schedule whereby a single lever press elicits the delivery of a food pellet to the receptacle. Only one lever is designated as “active” (triggering delivery of food reward) and the allocation of right and left levers is counterbalanced between mice. It is important to add at least a 5 second timeout (TO) to the FR1 schedule (FR1/TO-5), during which additional lever-pressing does not result in the delivery of a food pellet. This TO period allows time for mice to consume the food pellet, however longer time outs may be used if the experimenter feels it is necessary for their mouse model. Each FR training session lasts 1 hour or when 50 pellets have been delivered.
2. *Acquisition Criteria: The criteria used to determine acquisition of food-maintained operant responding includes: (1) a minimum number of active responses and rewards earned; (2) a measure of discrimination between active and inactive levers, and (3) between-session performance stability. Mice exhibiting discrimination of ≥3:1 for the active versus inactive lever and obtaining ≥ 20 rewards per session over three consecutive sessions are considered to achieve acquisition criteria¹⁰. We have observed that the majority (~75%) of C57BL6 mice require about 7-10 days of training to achieve acquisition criteria. Mice that do not reach acquisition criteria by this time undergo further training for an additional 5-7 days. We exclude mice from testing (~5% of cases) when there is no progress by the extra 5th training day. It is important to keep in mind that impaired operant acquisition may be an outcome of certain pharmacological and genetic interventions and should therefore be documented.
3. Following three successive sessions of obtaining ≥ 20 pellets, the schedule is increased to FR5/TO-5 seconds in which 5 active lever presses trigger the delivery of the food pellet. Training on the FR5 schedule lasts three days.
4. *The mice are then trained in the progressive ratio (PR) schedule of reinforcement. The response ratio schedule during PR testing can be calculated as per Richardson and Roberts (1996)¹¹ using the following formula (rounded to the nearest integer):
   = [5e ^(R*0.2)] – 5
   where R is equal to the number of food rewards already earned plus 1 (i.e., next reinforcer). Thus, the number of responses required to earn a food reward follow the order: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95 and so on. The final ratio completed is the breakpoint.
5. A PR session lasts a maximum of 1 hour. Failure to press the lever in any 10 min period results in termination of the session. Performance on the PR schedule of reinforcement is considered stable when the number of rewards earned in a 1 hour session deviates by ≤10% for at least 3 consecutive days.

4. Progressive Ratio Testing and Validation

1. To validate food-maintained operant responding in the PR task, one can assess breakpoint responding following manipulations known to modulate food reward. A relatively easy test to carry out is food deprivation which will increase the motivational state of the mouse for food and thereby increase breakpoints. Mice must first be tested in the PR task during a period of ad libitum feeding until stable, baseline performance is achieved. The day after the last “baseline” day food is removed from the home cage and mice are then tested in the PR task 24 hours after the start of the fast.
2. We also investigated whether peripheral administration of the anorectic hormone leptin would have the opposite effect on breakpoint thresholds. Leptin (A.F. Parlow, National Hormone and Peptide Program, NIDDK) was dissolved in sterile PBS. PR testing was carried out following an IP injection of PBS on one day and then following injection of leptin (5 mg/kg) the following day. Leptin was injected one hour prior to PR testing. Breakpoints were compared using a paired t-test (GraphPad Prism).

5. Representative Results

When testing the ability of food deprivation to modulate breakpoint responding on a PR schedule of reinforcement, we expect to significantly increase breakpoints as shown in Figure 1. In this experiment a 24 h period of food deprivation produced a ~3.6-fold increase in breakpoints as compared to those obtained in the free-feeding baseline state thereby suggesting that food deprivation increases the rewarding effects of food. In contrast, peripheral administration of the anorectic hormone leptin (5 mg/kg, IP) one hour prior to testing should decrease breakpoint responding as shown in Figure 2.

Figure 1. Fasting increases the rewarding effects of food. A 24 h fast significantly increased breakpoints on a progressive ratio (PR) schedule of reinforcement in mice (n=4). Mean±SEM; *p<.05.

Figure 2. Leptin decreases food reward in mice. Peripheral leptin administration (5 mg/kg) decreased PR breakpoint response thresholds in mice (n=4). Mean±SEM; *p<0.05.