This work was supported by the National Institute of General Medical Sciences (Reed/Engler-Chiurazzi - U54GM104942), the National Institute for Neurological Disorders and Stroke (Ashe &#8211; R01NS33249, R01NS63249 and R01NS79374), CoBRE (Engler-Chiurazzi - P20GM109098), the Alzheimer&#39;s Association (Reed - NIRG-12-242187), a WVU Faculty Research Senate Grant (Reed), a WVU PSCOR grant (Reed), and internal funds from the WVU College of Medicine Dean&#8217;s Office (Engler-Chiurazzi). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or Alzheimer&#39;s Association.

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The authors have nothing to disclose.

The MWM task is widely used to assess spatial learning and memory. However, the robustness of this task can be influenced by many factors and requires optimization for both background strain and testing environment. As shown in Figure 4, the same training protocol and applied visual cues used in two different testing rooms (equivalent size and layout) yielded significantly different probe performance. Since many features of the testing room might contribute to spatial cues 4, it was speculated...

Transgenic mouse models have been instrumental in evaluating the pathophysiology of Alzheimer&#8217;s disease (AD), as well as the potential of therapeutic interventions. Cognitive tasks, such as the Morris water maze (MWM), are commonly used with these models to identify the molecular correlates of memory deficits and to assess the efficacy of pre-clinical drugs. It is crucial, however, that the dynamic range of the cognitive task be wide enough to detect subtle treatment effects. With mouse models of AD, cognitive deficits are typically age-dependent, and mice display progressive declines in performance (e.g., 1). Use of a sensitive cognitive task can allow detection of subtle differences earlier in the animal&#8217;s life, thereby reducing the costs associated with aging animals. For example, reducing the number of training trials in the hippocampal-dependent Barnes maze from 15 to 5 increased the difficulty of the task, resulting in the detection of deficits in the 3xTg model at an earlier age than previously reported 2. Earlier detection of deficits not only offers considerable time and cost savings, it also increases the likelihood that the molecular changes underlying cognitive deficits can be identified.
One factor influencing the sensitivity of cognitive tasks is the genetic background strain of the mouse model. For example, BALB/c mice exhibit superior performance in learning and memory tasks compared to other strains, such as the C57BL/6 3. The F1 FVB/N x 129S6 background is used for two of the most widely employed models of AD, the Tg2576 and rTg(TauP301L)4510 models. This strain exhibits superior learning ability in the MWM relative to other strains, including B6/SJL mice 4. Because of this superior learning ability, the use of a single probe after extensive training may mask group differences resulting from over-training. In addition, the sensitivity of probe trials may be age-dependent. We have previously shown that earlier probe trials, after limited hidden platform training, are more sensitive to differences in young Tg2576 compared to young transgene-negative littermate controls than are probe trials inserted after more extensive training 5. In contrast, probe trials following extensive training are more sensitive in older (20-25 month) Tg2576 mice compared to older littermates than are earlier probe trials 5. By interspersing probe trials throughout training, the likelihood that a sensitive trial will be identified is increased, particularly if longitudinal testing is performed and the sensitivity of a particular probe trial is age-dependent. Figure 1 shows the superior performance of F1 FVB/N x 129S6 mice under the protocol optimized for this strain as compared to mice of the B6/SJL background trained under a protocol with more extensive training.
The MWM is generally thought to provide reliable measures that are reproducible across both time and laboratories 6. For example, the primary protocol originally used by our Minnesota laboratory 1,7 was successfully implemented with minor modifications at West Virginia University 8. Similarly, equivalent levels of impairment were observed in rTg(TauP301L)4510 mice relative to control littermates if housed under pathogen-free or conventional conditions 9. However, the testing environment can influence the sensitivity of the MWM task. Factors such as room lighting, air vents, temperature gradients, and noises all contribute to environmental cues 4 that can ultimately influence performance. When our Minnesota laboratory and vivarium were moved to a new building, up to a 38% reduction in wild-type performance was observed, substantially reducing the dynamic range of the task and the ability to detect transgene-related deficits. This change in performance occurred despite designing the testing room to be of equivalent size and configuration, and using the same applied visual cues. A &#8220;re-optimizing&#8221; of the original protocol was required to increase the dynamic range of the MWM task in the new testing environment.
Here the original protocol tailored for use with the F1 FVB/N x 129S6 background 5 is described. Because some studies suggest stress is associated with poor MWM performance 10 and pre-handling can alleviate this stress-induced deficit in performance 11, a pre-handling protocol was designed to acclimate the mice to the introduction and removal of the pool prior to MWM testing. Following pre-handling, mice undergo visible platform training, in which a raised platform is marked with a flag. Visible platform training is used to identify mice with performance problems related to sensorimotor abnormalities. Using exclusion criteria described in the protocol section, performance-incompetent mice are removed from subsequent examinations of hidden platform training and probe trials. Impairments in hidden platform training and probe trials are interpreted as cognitive deficits because sensorimotor performance is factored out of the data. After completion of visible platform training, mice begin hidden platform training where the platform is submerged in water and remains in the same position relative to external cues. Trials in which the platform is removed (probe trials) are interspersed throughout hidden platform training to assess the influence of additional training. Because probe trials occur at the beginning of each day, before additional hidden platform training, probe trials measure the ability of the animal to remember the location of the platform following a 20 hr delay, considered a measure of reference memory 12. Finally, ways in which this original protocol was re-optimized when changes in the testing environment disrupted control performance are described.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Viewer Tracking software</td>
			<td>Biobserve</td>
			<td></td>
			<td>This particular software is not a requirement - there are other tracking systems available</td>
		</tr>
		<tr>
			<td>Pre-handling pool</td>
			<td></td>
			<td></td>
			<td>Dimensions approximately 1 foot wide x 2 feet long x 1.5 feet deep</td>
		</tr>
		<tr>
			<td>Plastic beaker</td>
			<td></td>
			<td></td>
			<td>1 liter</td>
		</tr>
		<tr>
			<td>Scoop</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small net</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stopwatch</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>White circular tub</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Non-toxic white tempera paint</td>
			<td></td>
			<td></td>
			<td>Any color can paint can be used; must completely cover the hidden platform</td>
		</tr>
		<tr>
			<td>Platform</td>
			<td></td>
			<td></td>
			<td>Color should contrast that of maze</td>
		</tr>
		<tr>
			<td>Curtain rod</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curtains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse performance tracking software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Circular tub</td>
			<td></td>
			<td></td>
			<td>Uusally white in color; approximately 4 feet in diamater</td>
		</tr>
		<tr>
			<td>Platform</td>
			<td></td>
			<td></td>
			<td>Painted same color as the water</td>
		</tr>
	</tbody>
</table>

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.

We have used the Morris water maze to study the effects of beta-amyloid (Tg2576 mice) and mutant P301L tau (rTg(TauP301L)4510 mice) on spatial reference memory (e.g., 1,5,7,8). Figure 3 is the representative result reported in our study examining the effect of adult-onset P301L tau expression on learning and memory 8, utilizing testing Environment A. To assess motor and visual capabilities, mice were compared across visible platform training blocks, where each training bloc...

Watch this Scientific Journal Video about Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment at JoVE.com

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

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, ...

morris-water-maze-test-optimization-for-mouse-strain-testing

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Behavior

21.3K Views.  West Virginia University.  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. 

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

Shallow Water (Paddling) Variants of Water Maze Tests in Mice

When Richard Morris devised his water maze in 19817, most behavioral work was done in rats. However, the greater understanding of mouse genetics led to the mouse becoming increasingly important. But researchers found that some strains of mutant mice were prone to problems like passively floating or diving when they were tested in the Morris water maze11. This was unsurprising considering their natural habitat; rats swim naturally (classically, the "sewer rat"), whereas mice evolved in the dry areas of central Asia.
To overcome these problems, it was considered whether shallow water would be a sufficient stimulus to provide escape motivation for mice. This would also avoid the problems of drying the small creatures with a towel and then putting them in a heated recovery chamber to avoid hypothermia, which is a much more serious problem than with rats; the large ratio of surface area to volume of a mouse makes it particularly vulnerable to rapid heat loss.
Another consideration was whether a more natural escape strategy could be used, to facilitate learning. Since animals that fall into water and swim away from the safety of the shore are unlikely to pass on their genes, animals have evolved a natural tendency to swim to the edge of a body of water. The Morris water maze, however, requires them to swim to a hidden platform towards the center of the maze - exactly opposite to their evolved behavior. Therefore the paddling maze should incorporate escape to the edge of the apparatus. This feature, coupled with the use of relatively non-aversive shallow water, embodies the "Refinement" aspect of the "3 Rs" of Russell and Burch8.
Various types of maze design were tried; the common feature was that the water was always shallow (2 cm deep) and escape was via a tube piercing the transparent wall of the apparatus. Other tubes ("false exits") were also placed around the walls but these were blocked off. From the inside of the maze all false exits and the single true exit looked the same. Currently a dodecagonal (12-sided) maze is in use in Oxford, with 12 true/false exits set in the corners. In a recent development a transparent paddling Y-maze has been tested successfully.

Shallow Water Paddling Variants of Water Maze Tests in Mice

Mice can swim, but many strains appear to find this activity stressful. To overcome this problem mazes have been devised where escape from shallow water is used to motivate behaviour. These have been demonstrated to support learning at least as good as the traditional and widely used Morris water maze.

When Richard Morris devised his water maze in 19817, most behavioral work was done in rats. However, the greater understanding of mouse genetics led to ...

Use of an Eight-arm Radial Water Maze to Assess Working and Reference Memory Following Neonatal Brain Injury

Working and reference memory are commonly assessed using the land based radial arm maze.&#160;However, this paradigm requires pretraining, food deprivation, and may introduce scent cue confounds. The eight-arm radial water maze is designed to evaluate reference and working memory performance simultaneously by requiring subjects to use extra-maze cues to locate escape platforms and remedies the limitations observed in land based radial arm maze designs. Specifically, subjects are required to avoid the arms previously used for escape during each testing day (working memory) as well as avoid the fixed arms, which never contain escape platforms (reference memory). Re-entries into arms that have already been used for escape during a testing session (and thus the escape platform has been removed) and re-entries into reference memory arms are indicative of working memory deficits. Alternatively, first entries into reference memory arms are indicative of reference memory deficits. We used this maze to compare performance of rats with neonatal brain injury and sham controls following induction of hypoxia-ischemia and show significant deficits in both working and reference memory after eleven days of testing. This protocol could be easily modified to examine many other models of learning impairment.

The eight-arm radial water maze is designed to evaluate reference and working memory performance simultaneously by requiring subjects to use extra-maze cues to locate escape platforms and remedies the limitations observed in land based radial arm maze designs.

Working and reference memory are commonly assessed using the land based radial arm maze.&#160;However, this paradigm requires pretraining, food ...

Barnes Maze Testing Strategies with Small and Large Rodent Models

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.&#160;bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.&#160;distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.&#160;random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.&#160;shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.

The dry-land Barnes maze is widely used to measure spatial navigation ability in response to mildly aversive stimuli. Over consecutive days, performance (e.g.&#160;latency to locate escape cage) of control subjects improves, indicative of normal learning and memory. Differences between rats and mice necessitate apparatus and methodology changes that are detailed here.

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land ...

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 ...

A Protocol for Housing Mice in an Enriched Environment

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent studies demonstrate that environmental enrichment decreases adiposity, increases energy expenditure, resists diet induced obesity, and causes cancer remission and inhibition in mice. EE typically consists of larger living space, a variety of &#8216;toys&#8217; to interact with, running wheels, and can include a number of other novel environmental changes. All of this fosters a more complex social engagement, cognitive and physical stimulations. Importantly, the toy location and type of toy is changed regularly, which encourages the mice to adapt to a frequently changing and novel environment. Many variables can be manipulated in EE to promote health effects in mice. Thus these approaches are difficult to control and must be properly managed to successfully replicate the associated phenotypes. Therefore, the goal of this video is to demonstrate how EE is properly set up and maintained to assure a complex, challenging, and controlled environment so that other researchers can easily reproduce the protective effects of EE against obesity and cancer.

Environmental enrichment for mice requires a complex and challenging setup, as well as comprehensive husbandry and handling techniques to assure robust metabolic and anti-cancer effects in the mice. This protocol provides detailed procedures to reproduce the above mentioned effects in mice.

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent ...

Assessing Spatial Memory Impairment in a Mouse Model of Traumatic Brain Injury Using a Radial Water Tread Maze

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and validated for rat use. The Radial Water Tread (RWT) maze test of spatial memory (designed specifically for mice and requiring no swimming) has been shown previously to successfully distinguish between controlled cortical impact-induced TBI mice and sham controls. Here, a detailed protocol for this task is presented. The RWT maze capitalizes on the natural tendency of mice to avoid open areas in favor of hugging the sides of an apparatus (thigmotaxis). The walls of the maze are lined with nine escape holes placed above the floor of the apparatus, and mice are trained to use visual cues to locate the escape hole that leads out of the maze. The maze is filled with an inch of cold water, sufficient to motivate escape but not deep enough to require that the mouse swim. The acquisition period takes only four training days, with a test of memory retention on day five and a long-term memory test on day 12. The results reported here suggest that the RWT maze is a feasible alternative to rat-validated, swimming-based cognitive tests in the assessment of spatial memory deficits in mouse models of TBI.

Here we present a protocol for a mouse-specific test of cognition that does not require swimming. This test can be used to successfully distinguish controlled cortical impact-induced traumatic brain injury mice from sham controls.

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and ...

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 ...

Testing for Metacognitive Responding Using an Odor-based Delayed Match-to-Sample Test in Rats

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, numerous behavioral tasks have been created, many of which utilize an option to decline memory tests. To assess metamemory in rats, we utilized this decline-test option paradigm by adapting previous visual delayed-match-to-sample tests (DMTS)1,2 developed for primate species to an odor-based test suitable for rodents. First,&#160;rats are given a sample to remember by digging in a cup of scented sand. After a delay, the rat is presented with four distinctly scented cups, one of which contains the identical scent experienced during the sample; if this matching cup is selected, then the rat obtains a preferred, larger reward. Selection of any of the other three non-matching sand-filled scented cups results in no reward. Retention intervals are individually titrated such that subjects perform between 40 and 70% correct, therefore ensuring rats sometimes remember and sometimes forget the sample. Here, the operational definition of metamemory is the ability to distinguish between the presence and absence of memory through behavioral responding. Towards this end, on two-thirds of trials, a decline option is presented in addition to the four choice cups (choice trials). If the decline-test option- an unscented colored sand cup, is selected, the subject receives a smaller less-preferred reward and avoids the memory test. On the remaining third of trials, the decline-test option is not available (forced trials), causing subjects to guess the correct cup when the sample is forgotten. On choice tests, subjects that know when they remember should select the decline option when memory is weak rather than take the test and choose incorrectly. Therefore, significantly higher performance on chosen tests as compared to forced memory tests is indicative of the adaptive use of the decline-test response and metacognitive responding.

This protocol describes a method for investigating the possibility of metamemory, or memory awareness, in rodents. The odor-based delayed-matching-to-sample paradigm is a novel, ecologically-relevant behavioral test useful for determining the extent to which rodents can adaptively respond based on cognitively monitoring the strength of their memory states.

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, ...

Measuring Active and Passive Tameness Separately in Mice

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two behavioral components: a reluctance to avoid humans (passive tameness) and a motivation to approach humans (active tameness). To quantify these components in mice, we previously developed behavioral tests for active tameness, passive tameness, and the willingness to stay on a human hand, each designed to be completed within 3 min. The data obtained were used for selective breeding, with a large number of mice analyzed per generation. The active tameness test measures the movement of the mouse toward a human hand and the contact it engages in. The passive tameness test measures the duration of time that a mouse tolerates human touch. In the stay-on-hand test, a mouse is placed on a human hand and touched slowly using the thumb of that hand; the duration of time that the animal remains on the hand is measured. Here, we describe the test set-up and apparatus, explain the procedures, and discuss the appropriate data analysis. Finally, we explain how to interpret the results.

Tameness in animals includes a reduction of avoidance responses to humans (passive tameness) and an increase in active approaches to humans (active tameness). Here, we describe detailed protocols for three behavioral tests (active tameness, passive tameness, and stay-on-hand tests) to separately measure the active and passive tameness of mice.

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two ...

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.

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 ...