The aim of the following experiment is to train and test mice in a food motivated task using a progressive ratio schedule of reinforcement. This is achieved by first food restricting the mice to 60 to 70%of Ad Liam food intake to a stable reduction of five to 10%in body weight. To enhance the acquisition of food motivated lever pressing behavior, the mice are then trained to lever press for a high fat, high sugar food reward in the fixed ratio one, using an auto shaping procedure.
Miser then trained to respond for food on more complex schedules of reinforcement, including fixed ratio five, and then finally on the progressive ratio task, which provides a reliable measure of the rewarding effects of food. Next, after stable performance on the progressive ratio schedule, these mice are either food deprived for 12 to 24 hours or given intraperitoneal injection of retic agents to validate alterations in the food motivated behavior on the progressive ratio schedule of reinforcement. The results of this experiment show that food deprivation increases the food motivated behavior, whereas administration of the retic hormone leptin decreases the food motivated behavior for high fat and high sugar food rewards.
Upper conditioning tasks provide a useful means to evaluate the rewarding and motivational effects of different stimuli and behavior. The progressive ratio task is a well-validated schedule of reinforcement that has widely been used to assess the rewarding effect of drug of abuse and natural rewards such as food, although more commonly used in red studies application of the progressive ratio opera tasks to mice has become increasingly important with the growing generation of genetically engineered mouse models. In particular, the use of mouse models in obesity research and the recognition that the rewarding effects of palatable calorically rich food significantly contribute to overeating and obesity has emphasized the need to measure food reward in mice.
Here we explain and discuss the methods used to train and test mice to liver press for a palatable high fat, high sugar food on a progressive ratio schedule of reinforcement Before beginning. The protocol ensure that all procedures involving the use of animals have been approved by the institutional animal care. In use committee, it is recommended to test in the dark phase of the light dark cycle if needed.
Acclimate mice to a reverse light dark cycle for seven to 10 days. At this point, the mice are provided with ad libitum access to standard chow and water Following this 10 day acclimation period. The mice are singly housed and food restricted by providing 60 to 70%of ad libitum food intake until they reach 90 to 95%of their free feeding body weights.
This facilitates acquisition of leave press responding weigh the mice daily. Upon reaching five to 10%weight loss, the daily food allotted is adjusted to stabilize the lower body weights for the remainder of the training period three days prior to the first training session, transport the mice in their home cages to the testing room, then remove each mouse from its cage one at a time, and handle the mouse for two to three minutes. After handling, return the mouse to the home cage, acclimate the mice to the room and handling for three consecutive days the day prior to the start of training.
10 to 15 high fat high sugar pellets are placed into the home cage to prevent the potential influence of food. Neo phobia on operant performance, 20 milligrams of high fat, high sugar, dust-free precision food pellets containing 49%killer cow as fat are used here. Each mouse operant chamber is housed inside a sound attenuating cubicle with ventilating fan.
The mouse operant chambers are equipped with two ultra light retractable levers positioned on either side of the food receptacle. A stimulus light is located above each lever and there is a house light at the top of the cage. Appellate dispenser is located outside of the chamber.
The chambers are connected to a PC computer with Med PC four software via a smart control interface cabinet. Mice are introduced to the operant chamber. The initial training paradigm consists of a fixed ratio reinforcement schedule, one whereby a single lever press elicits the delivery of a food pellet into the receptacle.
Only one lever is designated as active and triggers the delivery of a food reward. The allocation of right and left levers is counterbalanced between mice. Training requires an also shaping procedure whereby crushed food pellets are placed on the lever to enhance lever pressing behavior.
During this initial training, an active lever response triggers delivery of the food reward and there is no delay between lever pressing and food delivery. Once mice have learned to press the lever a five second time, timeout is initiated during which additional leave of pressing does not result in the delivery of a pellet. The timeout period allows for mice to consume the pellet.
Longer timeout periods may be used if necessary after the timeout period pressing the active lever. Once again, results in the release of a food pellet each fixed ratio one five seconds. Timeout training session last one hour or when 50 pellets have been delivered.
Opera and training continues for seven to 10 days until acquisition of food maintained opera and responding mice exhibiting discrimination of equal to or greater than three to one for the active versus inactive lever and obtaining equal to or greater than 20 rewards per session. Over three consecutive sessions are considered to have achieved acquisition criteria. Mice that do not reach acquisition criteria by the seven to 10 day period undergo further training for an additional five to seven days.
Mice are excluded from testing when there is no progress by the fifth 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. Following three successive sessions of obtaining equal to or greater than 20 pellets, the schedule has increased to the FR five T oh five seconds protocol in which five active leave presses triggers the delivery of the food pellet.
Training on the FR five T oh five seconds schedule last about three to five days until responding is stable. The meister then trained in the progressive ratio schedule of reinforcement. The response ratio schedule during PR testing can be calculated as per Richardson and Roberts, where R is equal to the number of food rewards already earned, plus one.
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 break point. The PR session lasts a maximum of one hour failure to press the lever in any 10 minute 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 one hour session deviates by no more than 20%for at least three consecutive days to validate food maintained operant responding in the PR task, breakpoint responding following manipulations known to modulate food reward are assessed. A relatively easy test to carry out is food deprivation, which will increase the motivational state of the mouse for food and thereby increase break points.
Mice are first tested in the PR task during a period of ad liberton 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 12 to 24 hours after the start of the fast, or to investigate an opposite effect on breakpoint thresholds, one can test peripheral administration of the antic hormone. Leptin PR testing is carried out following an IP injection of PBS on one day and then after injection of five milligrams per kilogram of leptin on the following day.
Leptin is injected one hour prior to PR testing as seen here. Fasting increases the rewarding effects of food a 24 hour fast, significantly increased break points on a progressive ratio schedule of reinforcement in mice as seen here. Leptin decreases food reward in mice since peripheral leptin administration decreased breakpoint response thresholds While attempting this procedure, it's important to remember that acquisition of food maintain operant responding is significantly enhanced by regular training and by increasing the motivational state of the mouse by implementing a food restriction regimen.
In addition, ensuring that mice exhibit relatively stable responding on one reinforcement schedule before moving on to more complex schedules is key to minimizing variable performance during testing. Obesity and obesity related illnesses are a growing problem that has increased the urgency to identify the molecules and the neural pathways involved. The progressive ratio schedule is a valuable tool for measuring the impact of neurobiological, genetic, or pharmacological manipulations on the rewarding effects of the food.
