Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System

Summary

Presented here is a protocol to study depression-like and anhedonic behavior in rats. It combines two well-established behavioral methods, the sucrose preference and novelty-induced hypophagia tests, with an automated food and liquid intake monitoring system, to indirectly investigate rodent behavior using surrogate parameters.

Abstract

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral studies in animal models. In this protocol, to study depression-like and anhedonic behavior in rats, the established sucrose preference and novelty-induced hypophagia tests are combined with an automated food and liquid intake monitoring system. Prior to testing, in the sucrose preference paradigm, male rats are trained for at least 2 days to consume a sucrose solution in addition to tap water. During the test, rats are again exposed to water and sucrose solution. Consumption is registered every second by the automated system. The ratio of sucrose to total water intake (sucrose preference ratio) is a surrogate parameter for anhedonia. In the novelty-induced hypophagia test, male rats undergo a training period in which they are exposed to a palatable snack. During training, rodents show a stable baseline snack intake. On test day, the animals are transferred from home cages into a fresh, empty cage representing a novel unknown environment with access to the known palatable snack. The automated system records the total intake and its underlying microstructure (e.g., latency to approaching the snack), providing insight into anhedonic and anxious behaviors. The combination of these paradigms with an automated measuring system provides more detailed information, along with higher accuracy by reducing measuring errors. However, the tests use surrogate parameters and only depict depression and anhedonia in an indirect manner.

Introduction

On average, 4.4% of the world’s population is affected by depression. These account for 322 million people worldwide, an 18% increase compared to ten years ago¹. According to estimates by the World Health Organization, depression will be second in the ranking of Disability Adjusted Life Years in 2020². To address the rising prevalence of affective disorders and establish new interventional strategies, it is necessary to further study this behavior. Prior and in addition to examination in humans, animal studies are necessary.

Several models have been established to study components of depressive behavior (i.e., forced swim test, tail suspension test, sucrose preference test, and novelty-induced hypophagia)^3,4. The sucrose preference test (SPT) and novelty-induced hypophagia (NIH) can detect depression-like behavior in animals. These tests themselves do not induce a state of depression in rodents but depict acute changes in behavior. Both the SPT and the NIH assess a characteristic trait of depression known as anhedonia, which is the loss of interest in the following: rewarding activities, activities that were once enjoyed by the individual⁵, and one aspect of the complex phenomenon of processing and responding to reward⁶. Both tests study the response to a rewarding stimulus in the form of palatable food. The extent of consumption serves as a surrogate parameter for anhedonia^7,8,9.

The value of tests investigating anhedonia is strongly dependent on the accurate determination of consumption resulting from precise measurement of the substance’s weight. Conventionally, this measurement is conducted manually once before and once after the test. However, this is prone to erroneous measurements for several reasons. First, rodents tend to hoard food, meaning that they remove food without consuming it immediately then hide it in a safe place. Thus, this loss of food may be included in the calculation of total consumption. Second, rats spill food and water, resulting in weight loss without respective consumption. Third, unintentional loss of liquid occurs due to the handling of the bottles by inserting and removing them from cages.

In an approach to reduce these sources of error, we combined the two common tests assessing anhedonia (SPT^3,4 and NIH⁹) with measurement of food and water intake using an automated food and liquid intake monitoring system. This procedure allows accurate investigation of the consumption of palatable substances as well as provides information about the experience of pleasure in rats as a feature of depression-like behavior. The abovementioned errors associated with manual measurement are reduced by using different approaches, which are illustrated later in more detail.

To provide information about microstructure, the automated intake monitoring system used in this protocol¹⁰ weighs the food (±0.01 g) every second. Thus, a stable weight is documented as “not eating”, and an unstable weight as “eating”. A “bout” is defined as change in stable weight before and after an event. A meal consists of one or more bouts and its minimum size in rats was defined as 0.01 g. A meal is separated from another meal in rats by 15 min (standardized value). Thus, food intake is considered to be one meal when the bouts occurred within 15 min and the weight change is as equal to or greater than 0.01 g. Meal parameters assessed in this protocol include meal duration, time spent in meals, bout size, bout duration, time spent in bouts, latency to first bout, and number of bouts.

Protocol

Animal care and experimental procedures followed the specific institutional ethics guidelines and was approved by the state authority for animal research.

1. Operation of the automated monitoring system

NOTE: When operating the automated monitoring system, it is crucial to document every action in the comment box included in the software immediately prior to the action. The description should be typed into the comment box, and by pressing Save, it is saved with a specific timepoint. The timepoints are significant when analyzing the data, since the system records continuously, and the period of interest must be indicated for analysis.

1. Installing, using and maintaining the automated monitoring system
   NOTE: This protocol uses adult male Sprague Dawley rats weighing 250–300 g (~10 weeks old). It is recommended to house rats in groups during the acclimatization period. The environmental conditions should be controlled with the following parameters: 12 h/12 h dark/light cycle with lights on at 6:00 A.M., humidity of 45%–65%, and temperature of 21–23 °C, and ad libitum access to water and standard rodent diet. Daily handling enables the animals to become accustomed to the investigators.
   1. Separate the rats so that every animal has an individual cage. Ensure that every rat stays separated during the protocol.
   2. Fill the housing cages with regular bedding with a 1–2 cm thick layer. This (reduced) amount decreases the possibility of contamination of microbalances and hoppers with spillage, thereby reducing measuring errors. Add plastic tubes (e.g., a 20 cm long piece of a plastic drainpipe with diameter of 8 cm) and gnawing wood as enrichment, while omitting paper tissues to reduce measuring errors.
   3. Prepare the cages for the automated solid and liquid food intake measurement by attaching two closed cage mounts with microbalances to custom-made holes in the front side of the cages. Place two empty hoppers on the cage mounts, one for the chow and one for the bottle.
      NOTE: The microbalances are connected via cables to a recording system attached to a computer and the respective software is installed on the computer.
   4. To start recording, open “Monitor” and press “Start”, then choose a place to save the data.
   5. Using the calibration (press “Calibrate”) function of the automated intake monitoring system, calibrate every balance by removing the hoppers and placing two different gauged weights on the cage mounts with balances. Do this at regular time intervals (weekly is recommended).
   6. Fill one hopper completely with chow (~100 g) and remove chow pieces and crumbles that are too small in size. Fill water into the bottle (~100 mL) and place it into the other hopper.
   7. Document the position of food and water (e.g., balance 1: food animal 1, balance 2: water animal 1).
   8. Place the rat in the cage and open all gates of the cage mounts so that it can eat and drink ad libitum.
      NOTE: For an accurate measurement, it is necessary to maintain the balances and hoppers daily by cleaning gently with a brush from spillage and removing small food crumbs from the food container. This will greatly reduce erroneous measurement. Close all gates during the daily maintenance.
2. Accessing data after the experiments
   1. Search in the comment box for the beginning and end timepoints of a period (e.g., training, test) that needs to be analyzed.
   2. Click on “View data” on the software to open the Data Viewer.
   3. Insert the timepoints in the boxes below “Begin time” and “End time”. Press the square in the left upper corner indicating the balance that recorded the information.
   4. Click on “PSC Totals” to access the microstructure data. Press the button “Export PSC Table” to export the data.
      NOTE: To compare the microstructure of individual animals (e.g., unstressed vs. stressed) using automated monitoring, individual animals can be selected in the “Data viewer” by pressing the appropriate square in the left upper corner. The PSC Totals shows only the microstructure for the selected animal. Statistical analysis cannot be performed with the system. The data needs to be extracted into a spreadsheet program/analyzing software.

2. Implementation of the sucrose preference test

1. Conducting the training period
   NOTE: Prior to the test, animals must be accustomed to the availability of two bottles for liquids on hoppers through the gates, while food should be provided from the tops of cages (set-up is shown in Figure 1). This training period should last for at least 2 days. It is performed in the home cages in the room where animals are held.
   1. Close all the gates. Remove the water bottle and food container from the microbalances.
   2. Place pre-weighed food (~50 g) on the top of the cage and document its weight daily using a regular balance to assess daily food consumption. Refill, if necessary.
   3. Clean a bottle with clear water and refill with around 100 mL of water. Place it back on the hopper.
   4. Fill a second clean bottle with 100 mL of freshly made 1% sucrose solution. Place it on the hopper.
      NOTE: Mark the bottles carefully and document their locations (e.g., balance 1: water animal 1, balance 2: sucrose solution animal 1).
   5. Open all gates. Document the start of training in the monitoring system. Leave the gates open for 24 h, resulting in ad libitum access to both bottles. After 24 h, close the gates and document the end of the training. Data from the 24 h interval can be assessed using the automated monitoring system by inserting the “begin time” and “end time”. The procedures are the same when a 1 h test interval is assessed.
   6. Clean and refill the bottles every 24 h. Prepare fresh 1% sucrose solution daily. Switch the position of the water and sucrose solution bottle daily to avoid habituation effects.
      NOTE: Conduct the training in all animals at least 48 h until the preference ratios reache ~1. The sucrose preference ratio is assessed directly after training using the “Data viewer”. It is calculated as the ratio of sucrose intake to overall intake (water plus sucrose intake).
   7. 24 h before the test, remove the bottle with the sucrose solution so the rat has access to standard chow and water only.
   8. Prepare one fresh bottle filled with tap water and one filled with a 1% sucrose solution, both with ~100 mL.
   9. Prior to testing, close all gates.
   10. Remove the bottle filled with tap water from the hopper and place the two fresh bottles, one filled with tap water and one filled with a 1% sucrose solution, on the hopper.
   11. Open all gates, document the start of the test in the monitoring system. Leave the gates open for 60 min. Close the gates after 60 min and document the end of the test.
   12. Assess the data (e.g., the sucrose/total fluid intake ratio).
       NOTE: The test can be repeated several times with intervals of training (at least 2 days) in between.

3. Implementation of the novelty-induced hypophagia test

1. Conducting the training period
   NOTE: Prior to testing, a daily 30 min training period of 5 days is recommended (set-up is shown in Figure 2). The aim is to achieve a stable baseline of palatable snack intake before the experiment. It is performed in the home cages in the room where animals are held.
   1. Close all gates and remove the hopper with standard chow.
   2. Fill a fresh hopper with the palatable snack (~50 g). Insert the crackers carefully into the hopper to prevent crumbling. Place the hopper on the cage mount on top of the microbalance.
   3. Open the gates for 30 min so that the rat has ad libitum access to the snack and water. Document the beginning of training in the monitoring system.
      NOTE: The rat should have no access to standard chow during the training period.
   4. Close the gates after 30 min and document the end of training in the monitoring system. Replace the snack with standard chow.
   5. Repeat this daily until a 1) stable baseline palatable snack intake is achieved (e.g., 1.5–2.0 g/30 min) and 2) intake does not statistically differ between training days.
2. Performing the novelty-induced hypophagia test
   1. Prepare an empty, freshly cleaned cage without bedding or enrichment attached to the automated food intake monitoring system. Place a hopper with a bottle of tap water and hopper with a palatable snack on the cage mounts.
      NOTE: The novel cage should placed in the same room where the rats are held and training conducted. Keep the gates closed.
   2. Remove the rat from the home cage and place in the novel cage.
   3. Open all gates for 30 min. Document the beginning of testing in the monitoring system.
      NOTE: During the 30 min of access to the snack, the size of snack intake and underlying microstructure parameters (e.g., latency to first meal) are recorded using the automated food intake monitoring system.
   4. Close the gates after 30 min and document the end of testing. Place the rat back into the home cage.
      NOTE: The test can be repeated several times with intervals of training (at least 5 days) in between.

Results

To test data distribution, the Kolmogorov-Smirnov test was used. T-tests were used when data were normally distributed and Mann-Whitney-U test was used, if not. One-way ANOVA followed by Tukey post-hoc test was used for normally distributed multiple group comparison. One-way ANOVA followed by Dunn’s multiple comparison test was used in cases of non-normal distribution. Differences between groups were considered significant when p < 0.05.

The SPT was performed on naïve r...

Discussion

The sucrose preference and novelty-induced hypophagia tests are two established techniques for evaluating anhedonia in rats. Their combination with the automated food intake monitoring system allows for more detailed analysis in undisturbed rats and reduces erroneous measurement.

The incidence of errors is reduced by different approaches. First, to address the error occurring due to spillage, the gap between the food hopper and gate allows crumbs generated during gnawing to fall onto the integ...

Materials

Name	Company	Catalog Number	Comments
Assembly LH Cage Mount - RAT-FOOD - includes Stainless cage mount, hopper, blocker, coupling	Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA	BCMPRF01
Assembly LH Cage Mount unplugged - RAT - FOOD includes stainless steel cage mount, hopper, blocker, unplugged adapter, coupling	Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA	BCMUPRF01
cage w/ 2 openings - RAT - costum modified cage - includes cage top and standard water bottle	Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA	BCR02	single housing
Data collection Laptop Windows - Configured w/ BioDAQ Software	Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA	BLT003
enrichment (plastic tubes, gnawing wood)			distributed by the animal facility
HoneyMaid Graham Cracker Crumbs	Nabisco, East Hanover, NJ, USA	ASIN: B01COWTA98	palatable snack for NIH test
low vibration polymer rack	Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA	BRACKR
male Sprague Dawley rats	Envigo	Order Code: 002
Model #2210 32x Port BioDAQ Central Controller - includes cables, and calibration kit	Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA	BCC32_03
Peripheral sensor Controller - includes cable	Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA	BPSC01
SigmaStat 3.1	Systat Software, San Jose, CA, USA		statistical analysis
Stainless steel blocker	Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA	BBLKR
standard rodent diet with 10 kcal% fat	Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA	D12450B
sucrose powder	Roth	4621.1	for SPT