Name: sucrose preference and novelty-induced hypophagia tests in rats using an automated food intake monitoring system
Uploaded: 2020-05-08T13:00:00
Description: Watch this Scientific Journal Video about Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System at JoVE.com

This work was supported by funding of the German Research Foundation (STE 1765/3-2) and Charit&#233; University Funding (UFF 89/441-176, A.S.).

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 ...

<ol>
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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...

On average, 4.4% of the world&#8217;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 anhedonia7,8,9.
The value of tests investigating anhedonia is strongly dependent on the accurate determination of consumption resulting from precise measurement of the substance&#8217;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 (SPT3,4 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 (&#177;0.01 g) every second. Thus, a stable weight is documented as &#8220;not eating&#8221;, and an unstable weight as &#8220;eating&#8221;. A &#8220;bout&#8221; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Assembly LH Cage Mount - RAT-FOOD - includes Stainless cage mount, hopper, blocker, coupling</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BCMPRF01</td>
			<td></td>
		</tr>
		<tr>
			<td>Assembly LH Cage Mount unplugged - RAT - FOOD includes stainless steel cage mount, hopper, blocker, unplugged adapter, coupling</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BCMUPRF01</td>
			<td></td>
		</tr>
		<tr>
			<td>cage w/ 2 openings - RAT - costum modified cage - includes cage top and standard water bottle</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BCR02</td>
			<td>single housing</td>
		</tr>
		<tr>
			<td>Data collection Laptop Windows - Configured w/ BioDAQ Software</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BLT003</td>
			<td></td>
		</tr>
		<tr>
			<td>enrichment (plastic tubes, gnawing wood)</td>
			<td></td>
			<td></td>
			<td>distributed by the animal facility</td>
		</tr>
		<tr>
			<td>HoneyMaid Graham Cracker Crumbs</td>
			<td>Nabisco, East Hanover, NJ, USA</td>
			<td>ASIN: B01COWTA98</td>
			<td>palatable snack for NIH test</td>
		</tr>
		<tr>
			<td>low vibration polymer rack</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BRACKR</td>
			<td></td>
		</tr>
		<tr>
			<td>male Sprague Dawley rats</td>
			<td>Envigo</td>
			<td>Order Code: 002</td>
			<td></td>
		</tr>
		<tr>
			<td>Model #2210 32x Port BioDAQ Central Controller - includes cables, and calibration kit</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BCC32_03</td>
			<td></td>
		</tr>
		<tr>
			<td>Peripheral sensor Controller - includes cable</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BPSC01</td>
			<td></td>
		</tr>
		<tr>
			<td>SigmaStat 3.1</td>
			<td>Systat Software, San Jose, CA, USA</td>
			<td></td>
			<td>statistical analysis</td>
		</tr>
		<tr>
			<td>Stainless steel blocker</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BBLKR</td>
			<td></td>
		</tr>
		<tr>
			<td>standard rodent diet with 10 kcal% fat</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>D12450B</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose powder</td>
			<td>Roth</td>
			<td>4621.1</td>
			<td>for SPT</td>
		</tr>
	</tbody>
</table>

sucrose preference and novelty-induced hypophagia tests in rats using an automated food intake monitoring system

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.

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&#8217;s multiple comparison test was used in cases of non-normal distribution. Differences between groups were considered significant when p &#60; 0.05.
The SPT was performed on na&#239;ve r...

Watch this Scientific Journal Video about Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System at JoVE.com

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

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.

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral ...

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.

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