Published: January 7th, 2019
Here, we present a protocol to investigate changes in the anxiety level of rodent animal models. The elevated plus maze (EPM) test, used together with a video tracking software, provides a reliable method to document the effect of various potential anxiolytic treatments in preclinical laboratory scenarios.
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 goal of this article is to describe the methodology of the EPM test in combination with a video tracking software in order to monitor changes in anxiety-related behavior and novel treatments in laboratory rodent models. The EPM test is a relatively simple behavioral assessment method, which was developed for investigation of quantifying anxiety behavior levels and anxiety responses of rats after the application of drug treatments1. Indeed, it has been demonstrated that the EPM test is a widely used and effective behavioral assay for the investigation of changes in the anxiety levels of rodents1,2. The applicability of the EPM test in rodents (mainly rats and mice) is based on their proclivity toward enclosed, dark spaces (approach), an unconditioned fear of open spaces/heights (avoidance), and their high level of innate motivation to explore novel environments. Consequently, the EPM test is a well-established methodology based on an approach-avoidance conflict2,3.
The EPM is a plus-shaped apparatus consisting of four elevated arms, which has been described by Handley and Mithani4 (Figure 1), and consists of two opposite arms that are open to the surroundings (open arms), whereas the two closed opposite arms (closed arms) are equipped with walls. After treatment, if increased time is spent on the open arms and/or an increased number of open arm entries compared to control (untreated) animals is detected on the EPM, this indicates an anxiolytic effect2,3. The most robust avoidance response has been demonstrated in the first 5 min after the start (placement of the rats in the intersection of the four arms of the EPM) of the EPM assay5; therefore, any behavior after a treatment is commonly recorded for 5 min on the EPM. As additional measures of an anxiety level, the number of head dips, rears (vertical standing of the rodent on two hind legs), fecal boli, as well as total arm entries (spontaneous motor activity) and different postures (stretching or freezing), can also be recorded on the EPM2. Thus, multiple behavioral parameters can be compiled to provide a comprehensive assessment of anxiety-related behavior.
In order to increase the validity of the results, two to three behavioral assays are commonly used together, such as the light-dark choice test, the social interaction test, and the EPM test, to measure the anxiety levels of different animal models6. The EPM assay performed alone on rodents is also a suitable method to investigate the anxiolytic or anxiogenic effect of different drugs7. The EPM test is sensitive not only to benzodiazepine-type anxiolytics (e.g., diazepam)8, but also, among others, to amino acid, monoamine, peptidergic and nucleosidergic compounds (e.g., N-methyl-D-aspartate (NMDA) antagonist AP7, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist CNQX, µ-opioid receptor agonist morphine, NPY1 antagonist BIBP3226, substance P, ghrelin, oxytocin, serotonin receptor agonists and antagonists such as 8-OH-DPAT and WAY-100635, and β1-adrenergic antagonist betaxolol)9,10,11,12. Consequently, the EPM assay on rodents is a suitable and sensitive method to investigate the influence of different treatments that influence brain areas involved in the anxiolytic effect (e.g., the amygdala, hippocampus, and limbic areas) and mechanisms of action (e.g., the serotonergic, GABAergic, and adenosinergic system) implicated in anxiety2. The agents tested in these EPM studies include exogenous ketone supplements that alter brain signaling in subtle ways that may require a sensitive method to detect behavioral changes.
In this article, we describe the EPM test used in combination with a video tracking software, which helps to eliminate experimental bias and facilitates the collection and analysis of behavioral alterations in response to novel anxiolytic treatments.
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.
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.
In general, several commonly used tests, such as the light-dark choice test, the social interaction test, and the EPM test, are used to measure the anxiety level in different animal models. However, the EPM assay alone is a suitable method to investigate, for example, the effect of exogenous ketone supplements on rodents' anxiety levels18,20.
The main advantage of the EPM method is that it relies on the rodents' instinctive proclivity toward dark, enclosed spaces, in addition to the unconditioned fear of heights and avoidance of open spaces.On the other hand, other methods used to study anxiety-like behavior are based on the behavioral responses to certain noxious stimuli, such as electric shock, food/water deprivation, loud noises, and exposure to predator odor3. These tests usually result in a conditioned response, while the EPM also represents a more humane alternative. Furthermore, the EPM can be a useful tool to study the involvement of different brain regions (e.g., limbic regions, hippocampus) and the underlying mechanisms (e.g., GABA, glutamate, serotonin, adenosine) of anxiety behavior2.
When applying treatments that are quite stressful for the animals (e.g., the oral gavage), it is important that all animals are handled the same way and by the same person, especially when assessing potential, subtle anxiolytic effects. If possible, introduction of the drug/compound in drinking water or via a palatable 'treat' may be a preferred method. To ensure that the same amount is administered to each animal, an oral gavage can be used. Based on the pharmacokinetic properties of the compound, it is usually advisable to test the animals on the EPM within 1 hour after gavaging. When selecting experimental subjects, it is important to consider their strain, sex, estrus cycle, and age, as well as body weight, according to the objectives and test substances2. In regard to age, when designing EPM studies and interpreting data, it is important to consider that the percentage of open arm entries linearly increases with age21 and the aging-related changes in EPM behavior are strain-specific22.
When conducting an EPM test, there are potential problems that need to be addressed. Sometimes animals need to be excluded from the analysis due to outlier tendencies (e.g., the animal never leaves the area where it was placed, almost falls off the apparatus, is distracted by a noise or event outside of the apparatus). Further complications with EPM testing may include treatments which cause sedation or hyperactivity because these types of effects need to be assessed via EPM parameters.
It is important to expose animals to the EPM test only once because decreased activity on the open arms and a decreased total time spent on the central platform were demonstrated on the second (repeated) exposure of rodents compared to the first exposure on the EPM14,15. Therefore, a single exposure of rodents to the EPM test is strongly recommended. However, if there is a minimum of three weeks between the first and second exposure to the EPM and the EPM set-up is moved to another room (different environment), the animals may be investigated by the EPM test more than once2.
The EPM is available in different materials, sizes (e.g., for mouse or rat), and colors, which needs to be considered when choosing study subjects. It is important to keep in mind that the odors left by the previous animal on the apparatus may change the behavior of the subsequent animal. Therefore, we recommend using an EPM made of a material that is easy to clean, such as acrylic glass (not transparent), which does not retain odors after washing. Avoid EPM apparatus made of wood. Preferably, use a matte color that is different from the color of the animals tested on the EPM (e.g., black if white animals are tested). The better the contrast between the animal and the enclosure, the better the detection of the animal and the higher the reliability and precision of the results obtained (distance covered, speed, tracking). EPM apparatus made from matte gray material are useful with white, black, and white and black animals.
A further advantage of the video tracking system is that in addition to the EPM, it offers a flexible and easy way to set it up with a wide variety of behavioral tests, such as water maze, open-field, plus/radial arm/T-Y mazes, place preference, forced swimming, and tail suspension tests.
In summary, the goal of this article is to describe the EPM test used in combination with a video tracking software to collect and analyze behavioral alterations in response to novel anxiolytic treatments. The possible applications of the EPM include the prescreening of newly developed pharmacological agents for the treatment of anxiety-related disorders. In addition to the anxiolytic and anxiogenic agents, the behavioral effect of different hormones and drugs of abuse can also be investigated. The influence of aging and exposure to various stressors can also be assessed. This study has concluded that when proper steps are taken, the use of the EPM has proven to be a sensitive method to assess behavioral changes associated with ketone supplementation18,20.
D'Agostino, D.P., Kesl, S., Arnold, P. Compositions and Methods for Producing Elevated and Sustained Ketosis. International Patent # PCT/US2014/031237. University of South Florida.
Ari, C., D'Agostino, D.P., Exogenous ketone supplements for reducing anxiety-related behavior. Provisional patent #62289749. University of South Florida.
Dominic P. D'Agostino and Csilla Ari are co-owners of the company Ketone Technologies LLC.
These interests have been reviewed and managed by the University in accordance with its Institutional and Individual Conflict of Interest policies. All authors declare that there are no additional conflicts of interest.
This work was supported by an ONR Grant N000141310062 and a GLUT1D Foundation Grant #6143113500 (to Dominic P. D'Agostino), by the National Development Agency of Hungary (under Grant No. TIOP-1.3.1.-07/2-2F-2009-2008; to Zsolt Kovács) and by the Department of Veterans Affairs (to Mark Kindy). The authors wish to thank Quest Nutrition LLC for supporting ongoing research on this topic (to Csilla Ari).
|Elevated Plus Maze for mice and rats
|SMART Video Tracking Software
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