This work was supported by an ONR Grant N000141310062 and a GLUT1D Foundation Grant #6143113500 (to Dominic P. D&#39;Agostino), by the National Development Agency of Hungary (under Grant No. TIOP-1.3.1.-07/2-2F-2009-2008; to Zsolt Kov&#225;cs) 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).

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.
<ol>
	<li>Select animals for the experiment, considering the potential influencing factors, such as strain, sex, estrus cycle, and age, as well as body weight2.</li>
	<li>Based on the individual experiment, determine the number of animals per group for the test. 
	NOTE: The group size will be dependent on the effect size that is expected with the test treatment. Power analyses are generally done before the experiment is initiated to determine the minimum number of subjects to be included given the variability in animal&#8217;s responses in any given task, as well as the number of experimental groups/conditions.</li>
	<li>Design the experiment (in which a battery of different behavioral tests, such as the open-field test, EPM test, hole-board test, and forced-swimming test will be used) carefully. 
	NOTE: Pre-exposure of the rodents to a novel test environment (such as an open-field test) immediately before the EPM tests may change the behavior of the animals on the EPM1,2.</li>
	<li>Handle all animals in a similar way before the EPM test. 
	NOTE: It has been demonstrated that different stress factors, application of drugs (e.g., injections), shipping stress, and handling can change the behavior and behavioral responses of rodents on the EPM16. Thus, habituation of the animals to an animal house (e.g., after shipment, for 1 - 2 weeks before the EPM test), experimental conditions, and treatment procedures (e.g., gavaging) are necessary. It is also important that the handling of the rodents and any experience with prior stressors, particularly immediately before testing, is consistent across animals and treatment groups.</li>
	<li>Conduct the behavioral studies in nocturnal animals, such as rats and mice, using a reverse light cycle, so that the behavioral assessment can be performed when the animals are in their dark, active phase. 
	NOTE: The effects of different housing conditions and light cycle/circadian rhythms on behavior and their influence on EPM results were demonstrated previously17, since the animals&#8217; hormones are regulated by the light cycle.</li>
	<li>Use the same experimenters during the procedures and ask them to avoid perfume or soaps with a strong odor.</li>
	<li>Ask the experimenters not to talk near the animal during the experiment or move objects near the EPM environment. 
	NOTE: It is critically important that the observer makes minimal movements and no noise when collecting behavioral data.</li>
	<li>Clean the entire EPM after each trial to erase any smells of previous animals which might interfere with the exploration by the test animal.</li>
	<li>(Recommended) Handle the animals for several days before the EPM test (picking it up gently by the torso and holding it for a minute or two) to acclimate them to the experimenter.</li>
	<li>When placing the animals on the EPM, make sure to handle all animals in a consistent manner and place each rodent in the EPM in the same position facing the same arm (e.g., in the center facing the open arm away from the experimenter).</li>
</ol>
2. Application of Exogenous Ketone Supplements
<ol>
	<li>Measure the body weight of the animals before starting any treatments to determine the dosage calculation for the treatment (e.g., intragastric gavage).</li>
	<li>Familiarize the animals to the intragastric gavage method (adaptation period) using water by gavage for 5 d before ketone supplementation (standard rodent chow/standard diet [SD] + water gavage; e.g., 2.5 g/kg body weight of water/day). Exclude the use of any animal that does not adapt to the intragastric gavage method.</li>
	<li>Following the adaptation period, feed the animals chronically for 83 d and subchronically for 7 d with SD and gavage daily with either water (e.g., 5 g/kg body weight/day; control group: n = 8), ketone supplements such as ketone ester (KE; 1,3-butanediol-acetoacetate diester; e.g., 5 g/kg body weight/day; n = 8), ketone salt (KS; Na+/K+&#8210;beta-hydroxybutyrate [&#946;HB] mineral salt; e.g., 5 g/kg body weight/day; n = 8), or KS+ medium chain triglyceride (1:1 ratio, KSMCT; n = 8)18,19,20. 
	NOTE: The animals that received intragastric gavage were tested on the EPM 1 h after treatment. Rats fed with standard rodent chow and gavaged with water (excluding ketone supplementation) served as control groups.</li>
</ol>
3. Anxiety Assay
<ol>
	<li>EPM apparatus 
	<ol>
		<li>Use the same apparatus across a study to standardize results. The EPM is a plus-shaped apparatus, which consists of four arms (e.g., the arms may be 10 cm wide and 50 cm long): two opposite arms are opened, and the two closed opposite arms are equipped with high (e.g., 30 cm) walls. The apparatus is elevated above the floor (e.g., by 55 cm)2. 
		NOTE: The most commonly used parameters are the accumulated time spent in the open arms and the number of entries into the open arms; however, the time spent in the closed arms and center, and the number of entries into the closed arms and center are measured, as well as the distance traveled in each area.</li>
		<li>Light up the EPM by using indirect lighting (i.e., direct the light source toward the ceiling instead of directly illuminating the EPM apparatus) and ensure all four arms are similarly illuminated (without shadows, see Figure 2). 
		NOTE: Changes in the level of light alter the behavior of the rodents on the EPM. Therefore, similar illumination is needed in consecutive experimental animals and days (e.g., 2,800 lumens in the room)2.</li>
	</ol>
	</li>
	<li>Video tracking system 
	NOTE: Use a video tracking system with a computer interface and a video camera for the data collection, which will automatically collect behavioral data in rats (Figure 3). For the video tracking system, a wide variety of standard analog cameras or user-defined image sources (infrared cameras, camcorder, WIA-compliant USB camera, webcams, etc.) can be used. When analyzing the recorded video, the movement-tracking software supports all common video formats, such as .avi, .vob, .wmv, .asf, .mov, .qt, .mpg, .mpeg, .mp4, .3gp, and .mkv. If the video does not playback correctly, it might require a specific codec; additional video formats are supported if the corresponding codec is installed in the system. The movement-tracking software can also be used to analyze previously acquired videos and process the images from different sources, such as DVD/HD recorders, digital video files (.avi, .divx, .mpeg, etc.), webcams, DV cameras, and WIA-compatible imaging devices.
	<ol>
		<li>System setup
		<ol>
			<li>Plug the installation key of the movement-tracking software into a USB 2.0 port and launch the installation tool.</li>
			<li>Fix the camera above the experimental area and ensure that it will stay immobile for the duration of the experiment.</li>
			<li>Set up a new experiment in the movement-tracking software system by using the instruction manual. Select New Experiment. Double-click on the icon of the protocol that the new experiment should follow (Figure 3, Supplementary File 1).</li>
			<li>Enter details to label/describe the experiment in the Experiment Info dialog.</li>
			<li>Specify the source of the video sequences to process.</li>
			<li>Define the transformation rule for a correct distances measurement. The calibration process enables the movement-tracking software to be informed of the actual dimensions of the experimental area in order to obtain reliable values for distances and speeds.</li>
			<li>Determine the regions of interest (zones) in the working area.</li>
			<li>Adjust the parameters of the detection process.
			<ol>
				<li>In order for the movement-tracking software to precisely detect the position of the animal in the image, some detection adjustments must be set.</li>
				<li>The tracking process requires a clear and well-contrasted image by using a fine adjustment of the general brightness and contrast parameters in the Brightness &#38; Contrast section of the Detection Settings panel. As needed, adjust these settings for the whole image or for user-defined zones.</li>
			</ol>
			</li>
			<li>Put a rat into each arena to test the detection process.</li>
			<li>Press the Start Test button to verify if the detection process can identify the subject correctly. Confirm the detection is activated by the appearance of a dot on the screen. The calibration process has to be done before starting the test.</li>
			<li>Detection is considered confirmed when the only black dot shown in the player is the animal being tracked. The red tracking line needs to closely follow all the animal&#8217;s displacements. Proper tracking is also confirmed with a white label listing the animal number and corresponding coordinates based on displacement. If such a detection is not obtained, adjust the Threshold and Erosions parameters for optimizing the detection and tracking process.</li>
			<li>Adjust the Threshold and Erosion parameters to get a sharper and noise-free test image.</li>
			<li>If the tracking path is correctly detected, press the Stop Test button (Figure 4). If these adjustments are going to be used for every new experimental file, press the Save as default button. Press the Accept button to save the new detection settings.</li>
			<li>Set the time conditions of the trials.</li>
			<li>If the experimental protocol requires the track acquisition process to start at the same time the subject is placed into the experimental area, it is possible to set up the remote unit that comes with the software or to use a wireless mouse. 
			NOTE: This option provides the possibility of remotely controlling the start and stop.</li>
		</ol>
		</li>
		<li>Setup of the Subjects in the system
		<ol>
			<li>Manage the experimentation subject&#8217;s database. To create a database of experimental subjects, enter the Subjects Database manager by pressing the Subjects button in the Experimentation Assistant bar.</li>
			<li>Press the + button to add new subjects to the database.</li>
			<li>With the one subject option already selected, enter the subject&#8217;s code.</li>
			<li>Fill the rest of the subject&#8217;s information in the Subject Properties section.</li>
			<li>Press the Create button to add the new subject.</li>
			<li>Define the experimentation plan. Use the Scheduler to define the different phases, sessions, trials, and subjects planned to be executed within the experimental project. The trial is selected automatically as &#8220;the next trial&#8221; to be executed. This property is shown as a green tick on the left side of the trial name.</li>
		</ol>
		</li>
		<li>Data acquisition by simultaneous recording and tracking 
		NOTE: When a live image source is selected, the Player panel provides an embedded recording module for easily capturing the video coming from the selected camera.
		<ol>
			<li>Prepare the movement-tracking software for data acquisition (calibration, zone definition, detection settings, time settings, scheduler).</li>
			<li>Open the Data acquisition panel.</li>
			<li>Start recording the video of the experiment without the animal by pressing the Start recording button available in the software.</li>
			<li>Place the animal into the experimental area.</li>
			<li>Start the data acquisition process by pressing the Start button on the Time control panel. The tracking process will be carried out simultaneous with the recording process. As needed, ask the experimenter to note down the behavioral variables manually, such as rears, head dips, and falls (Figure 5).</li>
			<li>Collect the EPM data manually as well as by a blinded observer (separate the observer from the EPM by a curtain) in the testing room.</li>
			<li>Wait until the end of the tracking-process recording or press the Stop button on the Time control panel.</li>
			<li>Remove the animal from the experimental area. Stop the video-recording process by pressing the stop button available on the movement-tracking software player.</li>
			<li>Prepare the experimental area for the next animal by washing and drying it. Repeat the cycle again.</li>
		</ol>
		</li>
		<li>Data analysis 
		<ol>
			<li>To access the Analysis tool, press the Analysis button in the Experimentation Assistant bar.</li>
			<li>To generate analysis reports of the finished trials, select the trials to analyze. Configure and select the analysis report. Set the time intervals to be analyzed. Generate and review the reports. Export the results to a spreadsheet or image formats (Figure 6).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>EPM for the measurement of anxiety levels
	<ol>
		<li>Perform the EPM experiments under nonstress conditions (in a dimly lit and quiet room) after oral gavage. 
		NOTE: Make sure that the experiments are run in a close time interval (e.g., between 1200 and 1400) because the circadian rhythm can influence the rodents&#8217; behavior on the EPM15,17. Avoid unnecessary movements and noise during the experiment.</li>
		<li>Before the start of the test, make sure that the EPM is cleaned and dried and the video tracking system is ready to use.</li>
		<li>Transfer the rats in their home cage to the experimental room 30 min prior to beginning the experiment.</li>
		<li>Place a rat at the intersection of the four arms of the EPM, facing the open arm opposite of the experimenter.</li>
		<li>Run the video tracking software, as well as manually record the behavior of the animal, for 5 min.</li>
		<li>If the animal falls off the EPM, pick it up and place it back on the same point of the EPM where it fell off. Exclude the behavioral data of this animal from the analysis. 
		NOTE: A loud noise or movement may immobilize/freeze animals on open arms. If a loud noise is heard during the experiment, exclude the behavioral data of the animal undergoing the experiment at that moment from the analysis.</li>
		<li>At the end of the 5 min test, stop the video tracking software and remove the animal from the EPM. Place it back into its home cage.</li>
		<li>Before the next experiment/animal, clean the EPM with a disinfecting detergent (e.g., Quatricide) followed by tap water. Dry the apparatus with paper towels.</li>
	</ol>
	</li>
</ol>
4. Analyses of the Data Collected by the Video Tracking System
<ol>
	<li>Based on the recorded data, analyze the amount of time spent in the open arms and in the closed arms; the number of entries made to the open arms, closed arms, and to the center zone; the latency to entry into the closed arms; the distance traveled in the open arms, closed arms, and in the center zone. 
	NOTE: The animal is considered to be in an area when the center of the body mass is in that area.</li>
	<li>Determine the effects of treatments on behavior by using an analysis of variance (ANOVA) with Fisher&#8217;s least significant difference (LSD) test/Tukey&#8217;s multiple comparisons test.</li>
</ol>

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D&#39;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&#39;Agostino, D.P., Exogenous ketone supplements for reducing anxiety-related behavior. Provisional patent #62289749. University of South Florida.
Dominic P. D&#39;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.

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&#39; anxiety levels18,20.
The main advantage of the EPM method is that&#160;it relies on the rodents&#39; 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.,&#160;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 &#39;treat&#39; 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.

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, &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist CNQX, &#181;-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 &#946;1-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.

<table border="0" cellpadding="0" cellspacing="0" style="width:744px;" width="744">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Elevated Plus Maze for mice and rats</td>
			<td style="width:159px;">Coulbourn Instruments</td>
			<td style="width:133px;">&#160;H10-35-EPM</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SMART Video Tracking Software</td>
			<td>Harvard Apparatus</td>
			<td>SMART 3.0</td>
			<td style="width:236px;"></td>
		</tr>
	</tbody>
</table>

elevated plus maze test combined with video tracking software to investigate the anxiolytic effect of exogenous ketogenic supplements

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&#39; 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 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 &#177; the standard error of the mean (SEM). The results were considered significant when p &#60; 0.05. The significance was determined by one-way ANOVA with Fisher&#39;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, &#60; 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58396/58396fig1.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/58396/58396fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58396/58396fig2.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/58396/58396fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58396/58396fig3.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/58396/58396fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58396/58396fig4.jpg" /> 
Figure 4: The subject track is marked with a red line following the animal&#39;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. <a href="https://www.jove.com/files/ftp_upload/58396/58396fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58396/58396fig5.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/58396/58396fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58396/58396fig6.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/58396/58396fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58396/58396fig7.jpg" /> 
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&#160;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 &#60; 0.05; ** p &#60; 0.01; *** p &#60; 0.001; **** p &#60; 0.0001). This figure has been modified from Ari et al.18. <a href="https://www.jove.com/files/ftp_upload/58396/58396fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58396/58396fig8.jpg" /> 
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 &#60; 0.05; *** p &#60; 0.001; **** p &#60; 0.0001). This figure has been modified from Ari et al.18. <a href="https://www.jove.com/files/ftp_upload/58396/58396fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Elevated Plus Maze Test Combined with Video Tracking Software to Investigate the Anxiolytic Effect of Exogenous Ketogenic Supplements at JoVE.com

Elevated Plus Maze Test Combined with Video Tracking Software to Investigate the Anxiolytic Effect of Exogenous Ketogenic Supplements

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 ...

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30.2K Views.  University of South Florida. 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.

Video: Elevated Plus Maze Test Combined with Video Tracking Software to Investigate the Anxiolytic Effect of Exogenous Ketogenic Supplements

Mouse Short- and Long-term Locomotor Activity Analyzed by Video Tracking Software

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice and other rodents. Improvements in video tracking software (VTS) have allowed it to be coupled to LMA testing, dramatically improving specificity and sensitivity when compared to the line crossings method with manual scoring. In addition, VTS enables high-throughput experimentation. While similar to automated video tracking used for the open field test (OFT), LMA testing is unique in that it allows mice to remain in their home cage and does not utilize the anxiogenic stimulus of bright lighting during the active phase of the light-dark cycle. Traditionally, LMA has been used for short periods of time (mins), while longer movement studies (hrs-days) have often used implanted transmitters and biotelemetry. With the option of real-time tracking, long-, like short-term LMA testing, can now be conducted using videography. Long-term LMA testing requires a specialized, but easily constructed, cage so that food and water (which is usually positioned on the cage top) does not obstruct videography. Importantly, videography and VTS allows for the quantification of parameters, such as path of mouse movement, that are difficult or unfeasible to measure with line crossing and/or biotelemetry. In sum, LMA testing coupled to VTS affords a more complete description of mouse movement and the ability to examine locomotion over an extended period of time.

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice. Coupling of video tracking software (VTS) and LMA allows for the improvement of specificity and sensitivity, especially when compared with the manual, line crossing method of LMA analysis. Additionally VTS allows long-term tracking of mouse LMA.

Locomotor  activity (LMA) is a simple and easily performed measurement of behavior in mice  and other rodents. Improvements in video tracking software ...

A Dual Task Procedure Combined with Rapid Serial Visual Presentation to Test Attentional Blink for Nontargets

When viewers search for targets in a rapid serial visual presentation (RSVP) stream, if two targets are presented within about 500 msec of each other, the first target may be easy to spot but the second is likely to be missed. This phenomenon of attentional blink (AB) has been widely studied to probe the temporal capacity of attention for detecting visual targets. However, with the typical procedure of AB experiments, it is not possible to examine how the processing of non-target items in RSVP may be affected by attention. This paper describes a novel dual task procedure combined with RSVP to test effects of AB for nontargets at varied stimulus onset asynchronies (SOAs). In an exemplar experiment, a target category was first displayed, followed by a sequence of 8 nouns. If one of the nouns belonged to the target category, participants would respond &#8216;yes&#8217; at the end of the sequence, otherwise participants would respond &#8216;no&#8217;. Two 2-alternative forced choice memory tasks followed the response to determine if participants remembered the words immediately before or after the target, as well as a random word from another part of the sequence. In a second exemplar experiment, the same design was used, except that 1) the memory task was counterbalanced into two groups with SOAs of either 120 or 240 msec and 2) three memory tasks followed the sequence and tested remembrance for nontarget nouns in the sequence that could be anywhere from 3 items prior the target noun position to 3 items following the target noun position. Representative results from a previously published study demonstrate that our procedure can be used to examine divergent effects of attention that not only enhance targets but also suppress nontargets. Here we show results from a representative participant that replicated the previous finding.&#160;

This paper describes a novel dual task procedure combined with Rapid Serial Visual Presentation to look at attentional blink at varied stimulus onset asynchronies. This experimental procedure differed from others in that it tested attentional blink for nontargets.

When viewers search for targets in a rapid serial visual presentation (RSVP) stream, if two targets are presented within about 500 msec of each other, the ...

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g., allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.
Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; &#167;4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; &#167;4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.
Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.

The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their ...

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer&#8217;s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer's Disease

This protocol describes the administration of the 4 Mountains Test (4MT), a short test of spatial memory, in which memory for the topographical layout of four mountains within a computer-generated landscape is tested using a delayed match-to-sample paradigm. Allocentric spatial memory is assessed by altering the viewpoint, colors and textures between the initially presented and target images.
Allocentric spatial memory is a key function of the hippocampus, one of the earliest brain regions to be affected in Alzheimer&#39;s disease (AD) and impairment of hippocampal function predates the onset of dementia. It was hypothesized that performance on the 4MT would aid the diagnosis of predementia AD, which manifests clinically as Mild Cognitive Impairment (MCI).
The 4MT was applied to patients with MCI, stratified further based on cerebrospinal fluid (CSF) AD biomarker status (10 MCI biomarker positive, 9 MCI biomarker negative), and with mild AD dementia, as well as healthy controls. Comparator tests included tests of episodic memory and attention widely accepted as sensitive measures of early AD. Behavioral data were correlated with quantitative MRI measures of the hippocampus, precuneus and posterior cingulate gyrus.
4MT scores were significantly different between the two MCI groups (p = 0.001), with a test score of &#8804;8/15 associated with 100% sensitivity and 78% specificity for the classification of MCI with positive AD biomarkers, i.e., predementia AD. 4MT test scores correlated with hippocampal volume (r = 0.42) and cortical thickness of the precuneus (r = 0.55).
In conclusion, the 4MT is effective in identifying the early stages of AD. The short duration, easy application and scoring, and favorable psychometric properties of the 4MT fulfil the need for a simple but accurate diagnostic test for predementia AD.

The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer&#039;s Disease

This article describes the 4 Mountains Test (4MT), a hippocampus-dependent test of working allocentric spatial memory. The hippocampus is affected early in Alzheimer's disease (AD) and this article outlines the 4MT methodology and results of patient testing, which demonstrates the value of the 4MT in the diagnosis of pre-dementia AD.

This protocol describes the administration of the 4 Mountains Test (4MT), a short test of spatial memory, in which memory for the topographical layout of ...

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral science, conflicting outcomes are often provided from different laboratories. To identify reasons for the different outcomes, we previously focused on arm features, which differ between laboratories, most notably the presence/absence of ledges at the sides of open arms and the transparency/opaqueness of closed arm walls. In a previous report, we used a custom designed container to compare rat behavior on different combinations of open and closed arm designs under otherwise identical experimental conditions, and showed that differences in arm features interfere with experimental outcomes. In brief, open arm ledges significantly increased anxiety-like behavior in rats, while transparent arms may also have decreased this behavior. Furthermore, we verified a higher detection sensitivity of the effect of an anxiolytic drug in a combination of no-ledged open arm + opaque walled closed arm compared with a combination of ledged open arm + transparent walled closed arm. In this report, we introduce our protocol for the elevated plus maze test, together with discussion of the key results from the previous report and our experimental experience. We believe this report will provide useful information for researchers who have employed or who plan to use the elevated plus maze in their studies.

Rats' anxiety-like behavior tested by the elevated plus maze varies highly depending on the testing environment. Here we show how apparatus settings, especially arm structure, affect behavior on the maze, and we explain a reliable protocol to detect effects of anxiolytic drugs. This report will contribute to anxiety research.

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral ...

Utilizing the Modified T-Maze to Assess Functional Memory Outcomes After Cardiac Arrest

Background: Evaluating mild to moderate cognitive impairment in a global cerebral ischemia (i.e. cardiac arrest) model can be difficult due to poor locomotion after surgery. For example, rats who undergo surgical procedures and are subjected to the Morris water maze may not be able to swim, thus voiding the experiment.
New Method: We established a modified behavioral spontaneous alternation T-maze test. The major advantage of the modified T-maze protocol is its relatively simple design that is powerful enough to assess functional learning/memory after ischemia. Additionally, the data analysis is simple and straightforward. We used the T-maze to determine the rats&#39; learning/memory deficits both in the presence or absence of mild to moderate (6 min) asphyxial cardiac arrest (ACA). Rats have a natural tendency for exploration and will explore the alternate arms in the T-maze, whereas hippocampal-lesioned rats tend to adopt a side-preference resulting in decreased spontaneous alternation ratios, revealing the hippocampal-related functional learning/memory in the presence or absence of ACA.
Results: ACA groups have higher side-preference ratios and lower alternations as compared to control.
Comparison with Existing Method(s): The Morris water and Barnes maze are more prominent for assessing learning/memory function. However, the Morris water maze is more stressful than other mazes. The Barnes maze is widely used to measure reference (long-term) memory, while ACA-induced neurocognitive deficits are more closely related to working (short-term) memory.
Conclusions: We have developed a simple, yet effective strategy to delineate working (short-term) memory via the T-maze in our global cerebral ischemia model (ACA).

This protocol describes the use of a modified T-maze to evaluate functional learning/memory in asphyxia cardiac arrest-induced cerebral ischemia.

Background: Evaluating mild to moderate cognitive impairment in a global cerebral ischemia (i.e. cardiac arrest) model can be difficult due to poor ...

A Novel Single Animal Motor Function Tracking System Using Simple, Readily Available Software

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor deficits. Motor impairments were manually quantified through an open field grid test to measure the gross motor function and through a ladder test to measure the fine motor function. Here, we discuss a technique for the automated quantification of the video-recorded tests using our custom Capadona Behavioral Video Analysis System: Grid and Ladder Test, or BVAS. Leveraging simple and readily available coding software (see the Table of Materials), this program allows for the tracking of a single animal on both the open field grid and the ladder tests. In open field grid tracking, the code thresholds the video for intensity, tracks the position of the rat over the 3 min duration of the grid test, and analyzes the path. It then computes and returns measurements for the total distance traveled, the maximum velocity achieved, the number of left- and right-handed turns, and the total number of grid lines crossed by the rat. In ladder tracking, the code again thresholds the video for intensity, tracks the movement of the rat across the ladder, and returns calculated measurements including the time it took the rat to cross the ladder, the number of paw slips occurring below the plane of the ladder rungs, and the incidence of failures due to stagnation or reversals. We envision that the BVAS developed here can be employed for the analysis of motor function in a variety of applications, including many injury or disease models.

The current study aimed to automate the quantification of motor deficits in rats. The initial evaluation model assesses motor loss resulting from an intracortical microelectrode implantation in the motor cortex. We report on the development and use of a tracking algorithm using easily adaptable, simple, and readily available coding software.

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor ...

An Instrumented Pull Test to Characterize Postural Responses

Impairment of postural reflexes, termed postural instability, is a common and disabling deficit in Parkinson's disease. To assess postural reflexes, clinicians typically employ the pull test to grade corrective responses to a backward perturbation at the shoulders. However, the pull test is prone to issues with reliability and scaling (score/4). Here, we present an instrumented version of the pull test to more precisely quantify postural responses. Akin to the clinical test, pulls are manually administered except pull force is also recorded. Displacements of the trunk and feet are captured by a semi-portable motion tracking system. Raw data represent distance traveled (in millimeter units), making subsequent interpretation and analysis intuitive. The instrumented pull test also detects variabilities influencing pull test administration, such as pull force, thereby identifying and quantifying potential confounds that can be accounted for by statistical techniques. The instrumented pull test could have application in studies seeking to capture early abnormalities in postural responses, track postural instability over time, and detect responses to therapy.

Impairment of postural reflexes, termed postural instability, is difficult to quantify. Clinical assessments such as the pull test suffer issues with reliability and scaling. Here, we present an instrumented version of the pull test to objectively characterize postural responses.

Impairment of postural reflexes, termed postural instability, is a common and disabling deficit in Parkinson's disease. To assess postural reflexes, ...