The Sucrose Preference and Novelty-Induced Hypophagia Tests are established techniques for evaluating anhedonia. Their combination with the automated intake monitoring system, allows a detailed analysis with high accuracy. This protocol reduces the incidence of errors, by integrating spillage into the data analysis preventing harding, and reducing unintentional fluid loss.
Additionally, the protocol makes it possible to assess the intake microstructure. This technique aims to meet the need for valid tests, detecting anhedonia in rodents. Considering the rising prevalence of depressive disorders.
It can also be used to study antidepressive effects of drugs. When using the Automated Intake Monitoring System, it is important to remember, that it needs to be maintained and cleaned properly on a daily basis, in order to provide accurate data. To conduct the training, close all gates and remove the food container and water bottle, from the microbalances.
Place pre-weighed food on top of the cage and document its weight daily. Fill a clean water bottle with about 100 milliliters of water and place it back on the hopper. Fill a second bottle with 100 milliliters of freshly made 1%sucrose solution, and place it on the hopper.
Making sure to mark the bottles, and document their locations. Document the start of training in the monitoring system, and open all gates. Leave the gates open for 24 hours, resulting in ad libido maxes to both bottles.
Then document the end of training and close the gates. Prepare fresh sucrose solution daily and make sure to clean and refill bottles every 24 hours. Switching the position of the bottles to avoid habituation.
Conduct the training for at least 48 hours. Only proceeding with testing, When the sucrose preference ratios reach approximately one. 24 hours before the test, remove the bottle with the sucrose solution, so that the rat has access to standard chow and water only.
Prior to testing, prepare one fresh bottle filled with tap water and another filled with 1%sucrose. Close all gates. Remove the food container from the hopper and replace it with the prepared bottles of water and sucrose.
Then document the start of the test in the monitoring system and open all gates. Leave the gates open for 60 minutes. Then document the end of the test and close them.
To prepare for the training period, close all gates, and remove the hopper with the standard chow. Fill a fresh hopper with a palatable snack, inserting the crackers carefully to prevent crumbling. Then place the hopper on the cage mount on top of the microbalance.
Document the beginning of the training in the monitoring system and open the gates for 30 minutes so that the rat has ad libitum maxes to the snack and water. After 30 minutes, document the end of training and close the gates. Then replace the snack with standard chow.
Repeat the training daily until a stable baseline of palatable snack intake is achieved and does not statistically differ between training days. When ready to perform the test, prepare an empty freshly cleaned cage without bedding or enrichment and attach it to the automated food intake monitoring system. Place a hopper with a bottle of tap water and a hopper with a palatable snack on the cage mounts.
Remove the rat from the home cage and place it in the novel cage. Open the gates for 30 minutes. Making sure to document the start and end of testing.
When finished, place the rat back in its home cage. A Sucrose Preference Test administered to eight rats, showed an increase in consumption of sucrose and a decrease in water intake over the testing period. The Sucrose Preference Ratio increased from an average of 0.8 on the first day to 0.99 on the second day.
Sucrose intake microstructure was assessed automatically using the monitoring system. Meal size, meal duration, number of meals, and nine other parameters, were automatically measured. To demonstrate the advantages of using the Automated Intake Monitoring System, automated measurements were compared to those obtained, using conventional manual assessments.
All measurements were different between groups. Possibly due to erroneously high measurement or spillage when assessed manually. During the training period for the Novelty-Induced Hypophagia Test, overall intake of the palatable snacks steadily increased, indicating an adaptation during the first two to three days.
Similarly, meal size tended towards an increase between training days while meal duration did not. On the test day, naive rats exposed to the snack and a novel environment, showed a palatable snack intake of about one gram. Other parameters of food intake microstructure, such as meal duration, time spent in meals, and latency to first bout, were also assessed automatically.
When performing this protocol, document all the procedures, in a common section of the monitoring software, to save the time of the beginning, and the end of every session. Always document which substance is placed on which balance. Since this method is not associated with stress, the task can be repeated after an appropriate time of training in between testing.
After establishing this technique it was used to explore anhedonic features, of different Neuropeptides and undisturbed animals, expanding the knowledge of their functions.
