The animal treatment and measuring procedures were performed in accordance with the University of South Florida Institutional Animal Care and Use Committee (IACUC) guidelines (Protocol #0006R). All efforts were made to reduce the number of animals used.
1. Preparations
NOTE: The protocol typically requires laboratory-bred rats or mouse for EPM testing. However, other animals, such as guinea pigs, have also been tested on EPMs13. It is important to consider the color contrast between the animals in the maze and the maze color when using video tracking. The contrast is less important for researchers watching animals live or via video. The settings of the video tracking software need to be configured to document that the animals are black or white on either a black or white maze. Problems with configuration settings can occur with a clear acrylic maze, but a matte grey maze can be optimal for both rodent colors.
2. Application of Exogenous Ketone Supplements
3. Anxiety Assay
4. Analyses of the Data Collected by the Video Tracking System
The current experiment investigates the hypothesis that exogenous ketone supplementation administered either chronically (fed for 83 days) or subchronically (orally gavaged for 7 days) has an anxiolytic effect on two-month-old male Sprague-Dawley (SPD) rats (250 – 350 g). Chronic administration consisted of the following ketone supplements: low-dose ketone ester (LKE; 1,3- butanediol-acetoacetate diester, ~10 g/kg/day, LKE), high-dose ketone ester (HKE; ~25 g/kg/day, HKE), beta-hydroxybutyrate-mineral salt (bHB-S; ~25 g/kg/day, KS), and bHB-S + medium chain triglyceride (MCT; ~25 g/kg/day, KSMCT). For subchronic experiments, the following treatment groups were used: KE, KS, and KSMCT (5 g/kg/day). The control groups included SD or SD with water gavage (control). All data were represented as the mean ± the standard error of the mean (SEM). The results were considered significant when p < 0.05. The significance was determined by one-way ANOVA with Fisher's LSD test.
After chronic feeding, rats in the KSMCT group spent significantly more time in the open arms (p = 0.0094) compared to the control group. The time spent in the closed arms was significantly less in the LKE, KS, and KSMCT groups (p = 0.0389, 0.0077, and 0.0019, respectively), while the KS group spent significantly more time in the center (p = 0.0239) compared to the control (SD) group (Figure 7A)18.
Rats in the KS and KSMCT groups traveled significantly longer distances in the open arms (p = 0.036 and 0.0165), while the rats in the LKE, KS and KSMCT groups showed significantly less distance traveled in the closed arms (p = 0.0252, 0.00041, and 0.0032, respectively), compared to the control group (SD) (Figure 7B). When compared to the control group, the KS and KSMCT groups had greater distance traveled in the center area (p = 0.0206 and 0.0482, respectively), while in the KSMCT group, the latency to the first entrance to the closed arms was significantly greater after chronic feeding (p = 0.0038)18 (Figure 7C).
The time spent in the open arms was greater in the KE group (p = 0.0281) after 7 days of oral gavage, while in the KE, KS, and KSMCT groups, the time spent in the center decreased (p = 0.0005, < 0.0001, and = 0.023, respectively), compared to the control group (Figure 8A)18. In the KE and KS groups, the number of entries to the closed arms was significantly lower (p = 0.0436 and 0.0234, respectively) after 7 days of administration (Figure 8B), while the rats in the KS group also entered the center less frequently (p = 0.0193), compared to the control (SD) group.
Figure 1: Elevated plus maze (EPM) used for testing rats. Each arm is 10 cm wide and 50 cm long, with two opposite arms opened with a raised edge. The two closed opposite arms are equipped with 30 cm-high walls. The runway height from the floor is 55 cm. Please click here to view a larger version of this figure.
Figure 2: Examples of direct and indirect lighting. Ensure the light source is pointed toward the ceiling, while the direct light above the experimental area is blocked. It is important to use indirect light during EPM experiments in order to similarly illuminate all four arms without shadows. Please click here to view a larger version of this figure.
Figure 3: The experimentation assistant bar of the movement-tracking software. It is designed to provide access to the main operations. The buttons correspond to the task within the typical experimentation process, while only the currently allowed tasks are active. Please click here to view a larger version of this figure.
Figure 4: The subject track is marked with a red line following the animal's movement. By adjusting the threshold, the background can be decreased until only the animal is detected and tracked by the red line. The track is following the center of the mass of the subject, and the current position coordinates are indicated. Please click here to view a larger version of this figure.
Figure 5: Elevated plus maze (EPM) with a Sprague Dawley (SPD) rat in the open arm. An example of the experimental set-up is demonstrated. Please click here to view a larger version of this figure.
Figure 6: Accumulated movement track of the animal during a trial. As part of the data analysis, the collected trajectory trace of the subject in the tracking area can be displayed. Please click here to view a larger version of this figure.
Figure 7: Behavioral responses of SPD rats in the EPM after 83 days of chronic feeding of exogenous ketone supplementation. These panels show representative results collected by the EPM and the movement-tracking system18. (A) The KSMCT group spent a greater percentage of time in the open arms, while the LKE, KS, and KSMCT groups spent less time in closed arms, compared to the control (SD) group. (B) The KS and KSMCT groups traveled more distance in the open arms, while the LKE, KS, and KSMCT groups traveled less distance in the closed arms, showing reduced anxiety compared to the control (SD) group. (C) The KSMCT group entered the closed arms later, indicating reduced anxiety compared to the control (SD) group. Abbreviations: SD = standard rodent chow + water (25 g/kg body weight (b.w.) of water/day); LKE = SD + LKE (1,3-butanediol-acetoacetate diester, 10 g/kg b.w./day); HKE = SD + HKE (25 g/kg b.w./day); KS = SD + beta-hydroxybutyrate-mineral salt (bHB-S; 25 g/kg b.w./day); KSMCT = SD + bHB-S + medium chain triglyceride (MCT; 25 g/kg b.w./day); SPD = Sprague-Dawley rat; EPM = elevated plus maze (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). This figure has been modified from Ari et al.18. Please click here to view a larger version of this figure.
Figure 8: Behavioral responses of SPD rats after 7 days of oral gavage of exogenous ketone supplementation. Representative results were collected through the EPM test, using a movement-tracking software system18. (A) The KE group spent a greater percentage of time in the open arms, while the KE, KS, and KSMCT groups spent less time in the center (compared to the control [SD] group), thus indicating reduced anxiety. (B) Compared to the control (SD) group, less entries were detected in the closed arms from rats in the KE and KS groups. Abbreviations: SD = standard rodent chow + water (5 g/kg b.w. of water/day); KE = SD + ketone ester (1,3-butanediol-acetoacetate diester, 5 g/kg b.w./day); KS = SD + beta-hydroxybutyrate-mineral salt (bHB-S; 5 g/kg b.w./day); KSMCT = SD + bHB-S + MCT (5 g/kg b.w./day); SPD = Sprague-Dawley rat; EPM = elevated plus maze (* p < 0.05; *** p < 0.001; **** p < 0.0001). This figure has been modified from Ari et al.18. Please click here to view a larger version of this figure.
Elevated Plus Maze for mice and rats | Coulbourn Instruments | H10-35-EPM | |
SMART Video Tracking Software | Harvard Apparatus | SMART 3.0 |
The overall goal of this study is to describe the methodology of the elevated plus maze (EPM) test in combination with a video tracking software. The purpose of the method is to document the effect of various potential anxiolytic treatments on laboratory rodent models. The EPM test is based on the rodents' proclivity toward protected, enclosed dark spaces and unconditioned fear of open spaces and heights, and their innate intense motivation to explore novel environments. The EPM test is a widely used behavioral test for investigating the anxiolytic or anxiogenic responses of rodents given drugs that are known to affect behavior. Observation demonstrating a decreased proportion of time spent on closed arms, an increased proportion of time spent on open arms, a reduced number of entries to closed arms, and an elevated number of entries to open arms measured by the EPM test may reflect reduced anxiety levels. Using this method, the effect of exogenous ketone supplements on anxiety-related behavior is tested in Sprague Dawley (SPD) rats. Exogenous ketone supplements are chronically fed to the rats for 83 days or subchronically and acutely orally gavaged, daily for 7 days, before conducting the EPM test. Behavioral data collection is performed using the SMART video tracking system by a blinded observer at the end of the treatments. The main findings indicate that the EPM test is an effective method to detect the ketone supplement-induced anxiolytic effect and can be considered a sensitive measure to assess changes in anxiety behavior associated with drug- or metabolic-based therapies.
The overall goal of this study is to describe the methodology of the elevated plus maze (EPM) test in combination with a video tracking software. The purpose of the method is to document the effect of various potential anxiolytic treatments on laboratory rodent models. The EPM test is based on the rodents' proclivity toward protected, enclosed dark spaces and unconditioned fear of open spaces and heights, and their innate intense motivation to explore novel environments. The EPM test is a widely used behavioral test for investigating the anxiolytic or anxiogenic responses of rodents given drugs that are known to affect behavior. Observation demonstrating a decreased proportion of time spent on closed arms, an increased proportion of time spent on open arms, a reduced number of entries to closed arms, and an elevated number of entries to open arms measured by the EPM test may reflect reduced anxiety levels. Using this method, the effect of exogenous ketone supplements on anxiety-related behavior is tested in Sprague Dawley (SPD) rats. Exogenous ketone supplements are chronically fed to the rats for 83 days or subchronically and acutely orally gavaged, daily for 7 days, before conducting the EPM test. Behavioral data collection is performed using the SMART video tracking system by a blinded observer at the end of the treatments. The main findings indicate that the EPM test is an effective method to detect the ketone supplement-induced anxiolytic effect and can be considered a sensitive measure to assess changes in anxiety behavior associated with drug- or metabolic-based therapies.
The overall goal of this study is to describe the methodology of the elevated plus maze (EPM) test in combination with a video tracking software. The purpose of the method is to document the effect of various potential anxiolytic treatments on laboratory rodent models. The EPM test is based on the rodents' proclivity toward protected, enclosed dark spaces and unconditioned fear of open spaces and heights, and their innate intense motivation to explore novel environments. The EPM test is a widely used behavioral test for investigating the anxiolytic or anxiogenic responses of rodents given drugs that are known to affect behavior. Observation demonstrating a decreased proportion of time spent on closed arms, an increased proportion of time spent on open arms, a reduced number of entries to closed arms, and an elevated number of entries to open arms measured by the EPM test may reflect reduced anxiety levels. Using this method, the effect of exogenous ketone supplements on anxiety-related behavior is tested in Sprague Dawley (SPD) rats. Exogenous ketone supplements are chronically fed to the rats for 83 days or subchronically and acutely orally gavaged, daily for 7 days, before conducting the EPM test. Behavioral data collection is performed using the SMART video tracking system by a blinded observer at the end of the treatments. The main findings indicate that the EPM test is an effective method to detect the ketone supplement-induced anxiolytic effect and can be considered a sensitive measure to assess changes in anxiety behavior associated with drug- or metabolic-based therapies.