Documentation

Introduction

The Sucrose Preference Test Apparatus is a reward-based test used to investigate anhedonia in rodents. Anhedonia is the inability to feel pleasure in pleasurable activities or having a lack of interest in rewarding stimuli. In the Sucrose Preference Test Apparatus, the subjects are given access to two water bottles, one filled with sucrose solution, which serves as the rewarding stimuli and the other with regular water. Rodents have a natural preference for sweets; therefore, a reduced preference in the sucrose solution indicates the presence of anhedonia. 

The Sucrose Preference Test Apparatus consists of a black rectangular black box and allows numerous subjects to be tested at the same time. It has ten equal-sized chambers to place individual subjects. Inside each chamber, the two water bottles used for taste preference testing are placed. The chambers of the apparatus are separated by opaque black walls, which inhibit the subjects from seeing each other during trials to eliminate social interference.

The Sucrose Preference Test protocol is commonly used in combination with the Chronic Mild Stress Protocol in which the subjects are exposed to unpredictable mild stressors such as food and water deprivation to induce anhedonia (Gross & Pinhasov, 2016). The task involves testing the subjects in the Sucrose Preference Test Apparatus to obtain their baseline results, after which the subjects are exposed to a period of chronic mild stress. Sucrose preference testing is then repeated to observe whether depression-like behaviors are manifested in the subjects. Since the Sucrose Preference Test Apparatus is reliable in investigating anhedonia in subjects, it can effectively be used as a translational tool to measure the effects of antidepressant drugs in reducing depression-like behaviors, as well as explore the long-term neurobiological effects of antidepressant drug use (Flores-Ramirez et al., 2018). Moreover, the apparatus can also be used to test the effects of pharmacological manipulations, brain lesions, and diseases and disorders on depression-like behaviors. Other apparatuses used to measure depression-like behavior in rodents include the Tail Suspension Test, the Forced Swim Test, and the Chronic Social Defeat Stress Cage. 

Apparatus and equipment

The Sucrose Preference Test Apparatus consists of a rectangular box that is made of black polyethylene material that is 0.5 cm thick and measures 100 cm in length, 24 cm in width, and 13 cm in height. It is divided into ten equal-sized chambers by walls made of the same black polyethylene material. The chambers measure 10 cm in width, 24 cm in length, and 13 cm in height. Each chamber is covered by a transparent polyethylene cover that contains two 1-cm-diameter holes to allow the subjects to breathe. Inside each chamber, two liquid diet feeding tubes with holders are placed. The feeding tubes have a U-turn shape at their drinking end and are gastight to prevent leakage.

Training protocol

Clean the apparatus thoroughly before starting trials. Place a fresh tissue paper pad at the bottom of each chamber of the apparatus after every trial. Appropriately light the apparatus. A tracking and recording system such as the Noldus Ethovision XT can be used to assist with observations.

Habituation and Pre-training 

Place two water bottles in the home cages of the subjects, one containing sucrose (1% (wt/vol)) and one containing water for 48 hours. After 48 hours, in each chamber of the Sucrose Preference Test Apparatus, fill sucrose solution (1% (wt/vol)) in one of the bottles and fill regular water in the other bottle. Place a fresh tissue paper pad at the bottom of each chamber and place two food pellets on it. Place a subject in each chamber of the apparatus and allow them to access the sucrose solution and water for 24 hours. After 24 hours, return the subjects to their home cages.

Sucrose Preference Test Task 

Fill the two bottles in each chamber of the apparatus, one with sucrose solution (1% (wt/vol)) and the other with regular water. Dry the outside surface of the bottles and weigh them. Place a subject in each chamber of the apparatus and allow them to access the sucrose solution and water. After 12 hours, remove all the bottles from the chamber and weigh them. Return the subjects to their home cages for 12 hours. After 12 hours, perform another trial. After the second trial, place the subjects back into their home cages with access to regular food and water for 7 hours. After the 7 hours, remove the food and water from the subjects’ home cages and deprive them of food for 24 hours. Place the subjects back into the Sucrose Preference Test Apparatus and perform another trial.

Literature Review

Investigation of the effects of stress-induced anhedonia on sucrose preference in mice

Liu et al. (2018) investigated the effect of stress-induce anhedonia on taste preferences in mice using the Sucrose Preference Test Apparatus. Male and female mice of C57BL/6Ntac and ICR strains aged between 6 weeks and one year old were used in the experiment. The subjects were divided into control and experimental groups. The experimental group was exposed to a variety of chronic mild stress (CMS) to induce anhedonia for 28 days. Sucrose preference testing was performed before and after CMS induction. During sucrose testing, the two bottles in the chambers of the apparatus were each filled with either sucrose water (1% (wt/vol)) or regular water. The weight of the bottles was measured before and after every trial. After two trials, which served as baseline testing, the subjects were deprived of food and water for 24 hours. After deprivation, another trial of sucrose preference testing was performed. The results indicated no difference between control and experimental groups in sucrose preference testing before CMS. However, a difference in sucrose preference was seen between the two groups after CMS. Exposure to CMS caused a reduction in sucrose preference in the experimental group as compared to the control. The experiment was also repeated in mice that were treated with an antidepressant. Seven to eight-week-old male ICR mice were used in the experiment. The mice were divided into experimental and control groups. The experimental group was exposed to 56 days of CMS and administered with fluoxetine (10 mg/kg, i.p.) daily from day 22 to day 56, while the control group was treated with the same volume of saline only. Sucrose preference testing was performed from day 50. The results indicated that sucrose preference decreased after CMS; however, treatment with fluoxetine reversed these effects.

Investigation of the effect of nicotine withdrawal on sucrose preference in mice 

Alkhlaif, Bagdas, Jackson, Park, and Damaj (2017) investigated the effect of nicotine withdrawal on sucrose preference in eight-week-old C57BL/6J male mice. The subjects were divided into control and experimental groups. The control group was administered with saline, and the experimental group was administered with nicotine. Nicotine was administered subcutaneously for 14 days through mini osmotic pumps. The dose of nicotine was either 12 or 14 mg/kg/day, depending on the body weight of the subjects. On the 15th day, spontaneous nicotine withdrawal was conducted by the removal of the mini-pumps. Sucrose preference testing was performed for 11 consecutive days during nicotine exposure and nine consecutive days after the removal of the nicotine pumps. During sucrose preference testing, the subjects had access to two 30 ml tubes, one filled with water, and the other filled with a 2% sucrose solution. The measurements of sucrose and water intake were recorded every 24 hours. Results indicated a decrease in sucrose preference in nicotine withdrawal groups as compared to the saline group. The Light/dark box test was also performed to test the effect of nicotine withdrawal on anxiety-like behaviors. Results indicated that the nicotine withdrawn group significantly spent less time in the light compartment as compared to the saline group. The experiment was also performed using β2 and α6 nicotinic knockout (KO) and wild-type (WT) mice to investigate the role of β2 and α6 nicotinic subunits in nicotinic withdrawal behaviors. Sucrose preference testing was performed two days after the removal of the nicotine pumps. Results indicated a significant effect of genotype on sucrose preference behaviors. The nicotinic β2 WT and α6 WT mice had a decreased preference for sucrose as compared to their controls, unlike the β2 KO mice and the α6 KO mice. In the light/dark box test, the nicotinic β2 WT mice spent less time in the light compartment as compared to controls. However, similar to sucrose preference testing, nicotine withdrawal did not have a significant effect in β2 KO mice in the time spent in the light compartment as compared to controls. Nicotine withdrawn α6 WT mice also spent less time in the light compartment as compared to α6 KO mice. 

Investigation of the effect of chronic mild stress on sucrose preference behaviors in mice 

Gross and Pinhasov (2016) investigated the effect of chronic mild stress (CMS) on sucrose preference behaviors in submissive mice. Outbred Sabra (Wt) mice and submissive sabra (Sub) mice of 2 months old were used in the experiment to investigate whether the submissive mice would display heightened anxiety and depressive-like behavior relative to Wt after exposure to CMS. The subjects were divided into naïve and stressed groups. The naïve subjects remained in their social housing condition (4 mice per cage) while the stress group was transferred to individual housing. The subjects were further divided into control and CMS-exposed groups. Therefore, a total of 4 experimental groups were used in the experiment: naïve Wt, CMS-exposed Wt, naïve Sub, and CMS-exposed Sub. The CMS groups were exposed to daily random stressors for six days a week for five weeks. Sucrose preference testing was conducted on the days where the subjects were not exposed to stress. During sucrose preference testing, the subjects were given access to a bottle with a sucrose solution and a bottle with regular water. The subjects were exposed to 2.5% sucrose solution for 2 hours before starting trials to overcome neophobia. During testing, the subjects were exposed to 1% of sucrose solution or water for 24 hours. After testing, the subjects were divided into two groups in which the subjects were exposed to CMS or remained naïve. Results from the Wt group indicated that the five-week CMS exposure had an overall significant effect of time, stress, and time-of-stress interaction on sucrose consumption. CMS significantly reduced sucrose preference in the second and third week of testing. However, the effects of sucrose preference stabilized in the fourth and fifth weeks. In the Subgroup, similar results were observed. The subjects were also required to perform behavioral testing experiments on the Open Field Test Apparatus, the Elevated Plus Maze, and the Three Chamber Test. Behavioral testing was performed before and during the last week of CMS exposure. The results indicated that CMS exposure reduced locomotion in the open field test. In the elevated plus-maze, only the CMS-exposed Sub mice displayed impaired anxiety-like behavior. The CMS-exposed Sub mice also displayed impaired social exploration in the three-chamber test. 

Data Analysis

The following can be observed on the Sucrose Preference Test Apparatus:

• Weight of the first bottle before starting the trial
• Weight of the second bottle before starting the trial
• Weight of the first bottle at the end of the trial
• Weight of the second bottle at the end of the trial 

Strengths and Limitations

Strengths 

The Sucrose Preference Test Apparatus can be used to investigate anhedonia in rodents. It consists of a rectangular box that has ten chambers in which individual subjects can be placed. The multiple chambers allow numerous subjects to be tested at once, which can help save time. The chambers are divided by opaque black walls, which inhibit the subjects from seeing each other during trials to prevent social interference. Other fluids, such as saccharin, can also be used on the apparatus in place of sucrose. The Sucrose Preference Test Apparatus can effectively be used as a translational tool to test the effect of antidepressant drugs. The Sucrose Preference Test task is not too difficult or time-intensive. 

Limitations 

Factors such as age, gender, and strain of the subjects can affect task performance. Unintentional stimuli may interfere with task performance. It is important to randomly interchange the location of the two tubes to prevent the subjects from developing a bias towards one side. 

Summary

• The sucrose preference test apparatus is used to evaluate the presence of anhedonia in rodents and to distinguish anhedonia from depression. It can also be used to assess motivation and drug withdrawal. 

• Handling between training sessions can be potentially stressful for the subjects.

• Changes in the environmental parameters of the apparatus can affect the performance of the subject.

References

1. Alkhlaif, Y., Bagdas, D., Jackson, A., Park, A. J., & Damaj, I. M. (2017). Assessment of nicotine withdrawal-induced changes in sucrose preference in mice. Pharmacology, biochemistry, and behavior, 161, 47–52. https://doi.org/10.1016/j.pbb.2017.08.013
2. Flores-Ramirez, F. J., Garcia-Carachure, I., Sanchez, D. O., Gonzalez, C., Castillo, S. A., Arenivar, M. A., Themann, A., Lira, O., Rodriguez, M., Preciado-Piña, J., & Iñiguez, S. D. (2018). Fluoxetine exposure in adolescent and adult female mice decreases cocaine and sucrose preference later in life. Journal of psychopharmacology (Oxford, England), 269881118805488. Advance online publication. https://doi.org/10.1177/0269881118805488
3. Gross, M., & Pinhasov, A. (2016). Chronic mild stress in submissive mice: Marked polydipsia and social avoidance without hedonic deficit in the sucrose preference test. Behavioural brain research, 298(Pt B), 25–34. https://doi.org/10.1016/j.bbr.2015.10.049
4. Liu, M. Y., Yin, C. Y., Zhu, L. J., Zhu, X. H., Xu, C., Luo, C. X., Chen, H., Zhu, D. Y., & Zhou, Q. G. (2018). Sucrose preference test for measurement of stress-induced anhedonia in mice. Nature protocols, 13(7), 1686–1698. https://doi.org/10.1038/s41596-018-0011-z