This work was supported by an Ontario Mental Health Foundation grant to B.S, and an Ontario Graduate Scholarship to M.K.

All procedures are approved by the McMaster University Animal Care Committee and performed in accordance to guidelines set out by the Canadian Council of Animal Care.
1. General Procedures
<ol>
	<li>Habituate mice for 1-2 weeks to an existing 12 hr light/dark cycle (e.g., 8 AM &#8211; 8 PM). Perform all procedures and testing during the dark cycle, with RT, humidity and light intensity being relatively constant.</li>
	<li>Mark or tail-tattoo all mice for easy, numerical identification over a prolonged period and handle them 1-2 hr daily over 5-7 days.</li>
	<li>Repeat daily measurements of rectal temperature, body weight and food/water intake to detect potential fever and/or malnutrition induced by aging or disease progress. Standard exclusion criteria include low body weight due to reduced food/water intake, hunched posture with ruffled fur, hydrocephalus, porphyrin discharge around eyes, etc.</li>
	<li>To identify neurological deficits that may confound overall activity and performance, perform standard sensorimotor tests such as the hind limb clasping reflex 13, visual placing reflex 14, geotaxis test 15, basket test 16, beam walking test 17-19, Rotarod 20, and olfactory tests 21. 
	Note: The results may also help in correlational analysis with INBEST measures, more careful selection of other procedures (e.g., Morris water maze if mice are blind, novel object test if mice are hyposmic/anosmic), reduction of within-group variability, and exclusion of mice with birth deficits or infection.</li>
	<li>Clean the plastic and glass apparatus with disinfectant to remove urinary trails while testing mice from experimental and control groups in an alternating fashion.</li>
</ol>
2. Integrated Behavioral Station (INBEST) Procedure
<ol>
	<li>Home-cage set up
	<ol>
		<li>Fill food dispensers with 20 mg mouse chow pellets.</li>
		<li>Fill bottles with tap water. 
		Note: A second bottle can be filled with a solution of interest, such as sucrose or saccharine solution for a preference test.</li>
		<li>Weigh bottles to calculate the volume consumed at the end of the session.</li>
		<li>Insert bottle spouts into the lickometers. Make sure the nozzle does not block the infrared sensor; if it does, reduce the length of the spout.</li>
		<li>Place shelters in the selected corner of the home-cage.</li>
	</ol>
	</li>
	<li>Computer set up 
	Note: The software commands provided in steps 2.2-2.11 are relevant to the Ethovision XT 8.5 software package (specified in the List of Materials/Equipment) and testing conditions in our laboratory.
	<ol>
		<li>Illuminate the room with diffuse, dim light that is sufficient for video-tracking, but does not reflect off the box, floor or walls.</li>
		<li>Open a default video-tracking project and set up the Experiment Settings, by keying in relevant details (e.g., date/time of study, group assignment, room conditions etc.). Subsequently, choose the appropriate video source (Picolo Diligent grabber), number of arenas (4), tracking points (center-, nose- and tail-point), and units of measurement (centimeters, seconds, and degrees).</li>
		<li>After selecting the Trial List tab under Setup, define the number of trials by clicking the Add Trials button (1). Next, specify independent variables (e.g., mouse ID, gender, group assignment, strain) using the Add Variable button.</li>
		<li>Click the Arena Settings tab and capture the background image from the live video. Define the parameters of the individual arena by outlining the outer perimeter using the appropriate drawing tool (such as create rectangle/polyline/ellipse).</li>
		<li>Next, add zones of interest by clicking the Add Zone group button and outlining the various zones (e.g., floor, lickometers, food dispenser, climbing mesh etc.) in an identical manner. Add hidden zones for the areas where the mouse cannot be seen (e.g., shelter and running wheel, Figure 2) by clicking the Add Hidden Zone Group button. Ensure that an entry/exit is specified and linked to each hidden zone.</li>
		<li>Repeat steps 2.4-2.5 for each arena. Perform arena calibration by highlighting Calibration and using the appropriate tool (create calibration scale/axes) to provide arena width and length. Lastly, validate Arena settings by clicking the Validate Arena Settings button.</li>
		<li>Highlight the Trial Control Settings tab and specify start / stop conditions and trial length. Set the start condition to begin when the duration of the center-point exceeds 1 sec in the arena. Manipulate the trial duration by expanding the stop condition box and setting the trial to terminate after a delay, such as 10 hr.</li>
		<li>Under the Detection Settings tab, highlight the appropriate detection methods (e.g., dynamic subtraction and model-based).</li>
		<li>Next, grab the reference image of the empty arena by clicking the Settings button in the Detection tab and pressing the Grab Current button.</li>
		<li>Adjust the range of contrast such that the center, nose and tail-based detection for each mouse are reliable, accurate and continuous. For albino mice, specify that the mouse is brighter than the background, and darker than the background if using a pigmented strain. 
		Note: The subject size and video sample rate can be changed according to the distance between the overhead camera and the subject, as well as the processing speed of the PC used (e.g., 14.9 frames/sec).</li>
		<li>Ensure all changes are saved before exiting the detection settings module.</li>
		<li>Turn on the interface device, which is responsible for converting recorded analog events from input devices (e.g., interruption of infrared beam, movement of the running wheel etc.) into digital logs.</li>
	</ol>
	</li>
	<li>Data Acquisition 
	Note: The following software commands are relevant to the custom-made Med PC IV routine (&#8220;wizard&#8221;) that provides step-by-step input of session parameters (e.g., 10 hr trial duration, mouse ID, group assignment etc.).
	<ol>
		<li>Place each mouse into the assigned box.</li>
		<li>Synchronize video- and event-tracking packages by simultaneously pressing the &#39;record&#39; buttons.</li>
		<li>Leave the experimental room quietly.</li>
		<li>When the recording period expires (e.g., few hours, day, or weeks), remove mice and return them to their home-cages.</li>
		<li>Measure bottle weights and save all digital recordings on digital media (hard drive, portable USB stick, DVD).</li>
		<li>Transfer raw data to a spreadsheet.</li>
		<li>Save the MPG files for subsequent scoring of infrequent behavioral acts (e.g., stereotypy, seizures).</li>
	</ol>
	</li>
</ol>

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Open Access publication of this article is sponsored by Med Associates, Inc. (St. Albans, VT).

The detection of functional effects in animals largely depends on the researcher&#8217;s ability to limit the variability inherent to behavioral studies. Therefore, it is important to meticulously control and minimize potential confounds that may reduce the reliability and reproducibility of behavioral data. At the same time, it is important to recognize that no test reflects a single domain of behavior, that knowledge of neurological function is mandatory, and that behavior is highly sensitive to external stressors. If the above postulates are appreciated, one may conclude that comprehensive behavioral analysis should include the time course of the response measured, as well as, involve basic functional traits and paradigms that tap into specific behavioral aspects. Many of these criteria can be fulfilled using computerized assessment of movements and behavioral acts in an enriched home-cage environment.
Thus far, it has been emphasized that behavioral phenotyping of murine models of human disease warrants additional considerations. This notion is based on the premise that functional homeostasis is challenged by internal and external stressors during disease onset. Although all potential confounds may not be eliminated by the introduction of automated, home-cage phenotyping, issues relating to inconsistent environmental settings, transportation stress, and repeated handling are minimized. This significantly enhances consistency and precision across studies; even small reductions in variability can improve detection of the effects induced by an incipient disease. Indeed, INBEST provides a wealth of information, which permits more accurate assessment of the onset, kinetics, and severity of behavioral changes, as well as important relationships among various disease-induced behavioral deficits. Reliable video-tracking depends on two lighting conditions. First, diffuse light is required in the testing room to prevent artifacts from nearby reflecting objects. Second, high color contrast can be achieved by choosing an appropriate floor color to differ from the subject color as much as possible. In our lab, this is achieved by using flood-lights positioned below the INBEST boxes and black floor trays when monitoring albino mice (white or gray background would be adequate if testing pigmented strains). With respect to the event recording aspect of INBEST, the current hardware setting (1 Piccolo video card with 4 inputs) limits 4 boxes to be concurrently used per PC. This is a rather small number of boxes, while a more suitable set-up would require 8 or even 16 cages, and thus 2 or 4 PCs, respectively. Preferably, INBEST can be used continuously over 24-hr as the home-cage. This would allow animals to habituate fully to the environment and establish stable, circadian behavioral patterns, which can be analyzed in an unbiased manner. To prevent computer data loss due to power outage, a continuous power supply (or at least an uninterruptible power source) must be secured. Lastly, to ensure proper assessment of daily food intake, it should be noted that the size of food pellets should not exceed the size of the holes in the food dispenser (the recommended size of a single food pellet is 20 mg).
It is not to be overlooked, however, that this analysis should also incorporate how different INBEST measures may interact with each other. For example, mice that spend more time in the running wheel are likely to ingest higher quantities of food and water to meet their increased caloric demands. Similarly, mice ingesting more sucrose solution may decrease their intake of food. The interpretation of these results may further be complicated by the general improvement of performance over time, particularly in regards to ingestive behavior and running wheel activity. Given their incentive-properties, experimenters may also consider limiting access to sucrose solution and the running wheel to counteract the likelihood of post-ingestive effects and excessive weight loss, respectively. However, these concerns may be more relevant in some strains than others because different strains of mice have dissimilar behavioral profiles. Although performing both baseline and experimental assessment controls for many of the issues above, experimenters need to recognize that these variables need to be taken into account when interpreting INBEST data. At the same time, some aspects of behavior cannot be studied in the home-cage environment, thus necessitating a combination with standard tests to complete the behavioral profile of subjects.
Computerized monitoring within standardized, but flexible environments appears to be the logical next step in contemporary behavioral analysis. Such a non-invasive, ethologically-based approach will allow researchers to observe the full repertoire of behavioral responses over a prolonged period of time. Theoretically, this can be accomplished by studying behavior in a &#8220;virtual&#8221;, enriched environment that closely resembles a natural habitat. Several research groups have described vision-based tracking tools that support behavioral phenotyping of mice in their home-cage 22-25, in dyads 26, 27, or in the context of large social groups 28. High precision and spatial resolution can be achieved by integrating video-tracking with microchip technology for simultaneous and synchronized collection of behavioral data in a group of mice 28. Thermographic cameras capable of detecting heat-signatures can be combined with implantable microchips or transponders to provide the relative location and basic physiological functions of each mouse (e.g., body temperature, heart/breathing rate). In addition, an advanced 3D tracking system would produce more accurate and quantitative recognition of behavioral acts. In order to repeatedly run a variety of tests, such a system should be automated, remotely-controlled, and modular. For example, spatial memory can be studied in larger environments by programming the appearance of distal cues on LCD walls, or by presenting/hiding dispensers with palatable food from movable floors. In a similar manner, novel objects could be presented/hidden at specific times throughout the study. Such computerized phenotyping may help in elucidating the genetic determinants of behavior, pathogenic mechanisms underlying disease models, and the development of novel therapeutic strategies. If a consensus is reached with respect to testing conditions, sequence of tests, as well as the hardware and software used, one may expect that long-awaited standardization would improve reproducibility of behavioral studies and elevate experimental psychometrics to a new level.

Rapid advancements in genetic engineering over the past few decades have led to an unprecedented proliferation of animal models of human diseases. Mice have gained the status of primary experimental subjects in biomedical sciences for several reasons. From a practical standpoint, they have a high reproduction rate, are relatively inexpensive, and are easy to house. From a conceptual point of view, they are genetically close to humans, can be genetically modified with relative ease, and have highly-developed endocrine, immune, and nervous systems. In addition to the lesions at genetic and cellular levels, contemporary studies of brain disorders require the demonstration of replicable functional deficits that highlight face, construct, or predictive validity of a new mouse model 1.
An acute infection in a homoeothermic mammal often results in the febrile response, which together with sickness behavior, constitutes one of the principal survival mechanisms 2. Acutely sick animals display significant alterations in food/water intake and performance in tasks reflective of emotional reactivity, exploratory behavior, and learning/memory capacity. These changes largely account for impaired social/sexual activity and preservation of energy for defensive immune reactions. However, when acute conditions turn chronic (as seen in many immunological, endocrine and neurological diseases), behavioral performance can further deteriorate due to structural damage of various organs, including the brain 3.
Human and animal neurodegenerative diseases are often accompanied by a constellation of neurological and behavioral deficits. Therefore, a key purpose in behavioral studies of chronically sick animals is to distinguish central effects from the deficits induced by peripheral symptomatology. However, the relatively short duration of standard behavioral tasks limits the collection of information concerning basic functional measures, such as olfaction, resting, sleep, food/water intake, or epileptic episodes. Inclusion of these measures improves behavioral profiling and permits better interpretation of performance in activity-demanding tasks.
Refinements in behavioral phenotyping of diseased mice
The inadequacies in assessing the behavioral profile of sick mice have necessitated continuous monitoring of singly-housed mice by fast-processing PCs. Although diverse behavioral batteries can be designed 4, 5, listed below are the procedures that have been used to successfully establish an animal model of neuropsychiatric lupus 6. This battery is repeatedly applied in both sub-chronic and chronic models of disease (Figure 1), such as mild cognitive impairment and Alzheimer&#8217;s disease 7. Following a series of neurological tests 8-10, a custom-made apparatus, designed to meet the above demands by utilizing continuous monitoring of multiple behavioral outputs in an enriched home-cage-like environment, can be employed. Such an ethologically-based approach to the assessment of spontaneous exploratory activity and motivated behavior provides a more comprehensive understanding of performance deficits in other paradigms, such as those reflective of learning and memory.
<img alt="Figure 1" src="/files/ftp_upload/51524/51524fig1.jpg" /> 
Figure 1. Schematic representation of longitudinal behavioral phenotyping in our laboratory. The behavioral battery is designed to evolve from less- towards more- stressful tasks, which are repeated at different time points to assess the effects of sustained factors such as disease progression, pharmacological treatment, or immunological responses. INBEST and individual tests are performed during the dark phase, often over 10 and 2 hr, respectively. Abbreviations: R &#8211; reflexes; BW &#8211; Beam Walking test; RR &#8211; Rotarod; OT &#8211; Olfactory tests; SP &#8211; Sucrose Preference test; SD &#8211; Step Down test; NO &#8211; Novel Object test; OF &#8211; Open-field test; SAB &#8211; Spontaneous Alternation Behavior; FS &#8211; Forced Swim test; MWM &#8211; Morris Water Maze. * - aspects of the test (e.g., location, context, color, shape) that need to be altered in subsequent trials throughout course of experiment.
Continuous video-recording and analysis of behavior in a home-cage-like environment were first reported in 2007 11. A more complex automated apparatus that integrates behavioral tests used in studies with autoimmune mice was presented at the &#39;Measuring Behavior&#39; meeting one year later 12. The Integrated Behavioral Station (INBEST, Figure 2A) is a modular system, which comprises of a shelter, computer controlled light stimulus, two photocell-controlled lickometers (one for water, one for a solution of interest), an automated food dispenser, a computerized running wheel, and a digitized climbing mesh. Latencies, frequencies, and durations of specific behaviors are examined using customized software. Locomotor and exploratory activity (e.g., of a novel object or an unfamiliar conspecific) can be assessed with video-tracking software (List of Materials/Equipment), while sleeping and less frequent behavioral patterns, such as self-injurious behavior and seizures, can be scored manually with video-tracking software or dedicated event-recording packages. Eight complete INBEST/video setups are used, thus allowing concurrent monitoring of 4 experimental and 4 control animals (Figure 2B).
<img alt="Figure 2A" src="/files/ftp_upload/51524/51524fig2A.jpg" /> 
<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51524/51524fig2Ahighres.jpg" target="_blank">Please click here to view a larger version of this figure.</a> 
<img alt="Figure 2B" src="/files/ftp_upload/51524/51524fig2B.jpg" /> 
Figure 2. Integrated Behavioral Station. (A) Schematic representation of hardware, and software used in designing an INBEST box (L = 39 x W = 53 x H = 50 cm). (B) Eight complete INBEST boxes provide an opportunity for concurrent home-cage monitoring of four experimental and four control mice.
Dependent variables include measurements of food/water intake, responsiveness to palatable stimulation, spontaneous ambulatory activity, climbing, voluntary running, anxiety-related behaviors (e.g., exploration of novel object), grooming, seizing and sleeping. Additionally, visual stimuli can be presented for conditioning and learning paradigms. The advantages of INBEST over standard behavioral testing include the elimination of confounding effects induced by transportation stress, as well as continuous, automated collection of measures reflective of nocturnal activity, exploration, anxiety-related and depressive-like behaviors. The integration of sensitive hardware components with a video-tracking package yields a wealth of information, which permits improved assessment of behavior in relation to progression of chronic disease in diverse animal models. INBEST can be used to study other chronic CNS disorders (e.g., autism, major depression, schizophrenia), as well as in longitudinal studies focusing on neurodevelopment, behavioral effects of systemic / neoplastic disorders, and prolonged pharmacotherapy.

<table><tbody><tr><td>Power control interface operating package</td><td>Med Associates Inc.</td><td>MED-SYST-8</td><td>Interface box and PCI card that manage all A/D data inputs and outputs</td></tr><tr><td>Stimulus light</td><td>Med Associates Inc.</td><td>ENV-221M</td><td>28 V DC, 100 mA, 2.5&amp;nbsp;cm diameter light (for presentation of a conditioned stimulus)</td></tr><tr><td>Head entry detector</td><td>Med Associates Inc.</td><td>ENV-254-CB</td><td>Permits head entry detection into the pellet receptacle</td></tr><tr><td>Photobeam lickometer</td><td>Med Associates Inc.</td><td>ENV-351W</td><td>Infrared sensor system for detecting beam interception by snout</td></tr><tr><td>Food pellets</td><td>Bio-Serv</td><td>F0163</td><td>Dustless precisions food pellets (20 mg rodent grain-based diet)</td></tr><tr><td>Food dispenser</td><td>Med Associates Inc.</td><td>ENV-203-20</td><td>Automated food dispensing system consisting of elevated plastic container and dispensing tube</td></tr><tr><td>Food receptacle</td><td>Med Associates Inc.</td><td>ENV-303R2W</td><td>Infrared sensitive base to signal when food pellet is dispensed or collected</td></tr><tr><td>Climbing mesh</td><td>Med Associates Inc.</td><td>CT-Climbing mesh</td><td>Durable metal rungs, dimensions</td></tr><tr><td>Med PC IV software</td><td>Med Associates Inc.</td><td>SOF-735</td><td>Integrates data acquisition from all electronic devices</td></tr><tr><td>MPC2XL v1.4</td><td>Med Associates Inc.</td><td>SOF-731</td><td>Raw data transfer utility</td></tr><tr><td>Soft CR Pro v1.05</td><td>Med Associates Inc.</td><td>SOF-722</td><td>Remote online monitoring software</td></tr><tr><td>Running wheel</td><td>Med Associates Inc.</td><td>CT-MSUB-ENV-3042-X1</td><td>Activity wheel for mice</td></tr><tr><td>Digital counter</td><td>Med Associates Inc.</td><td>ESUB-ENV-3000</td><td>LCD counter (4 counts = 1 revolution = 54.6 cm length)</td></tr><tr><td>Picolo Diligent frame grabber</td><td>Euresys</td><td></td><td>High-resolution PCI video capture card</td></tr><tr><td>Ethovision XT 8.5</td><td>Noldus Information Technology</td><td></td><td>Video-tracking software</td></tr><tr><td>Camera</td><td>Panasonic</td><td>WV-BP334</td><td>Digital, low-lux video camera suspended from a custom-made metal stand</td></tr><tr><td>Video Splitter</td><td>American Dynamics</td><td>ADQUAD87</td><td>Integrates and digitizes inputs from 4 video cameras</td></tr></tbody></table>

behavioral phenotyping of murine disease models with the integrated behavioral station (inbest)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of chronic CNS disorders, there is an increasing demand for murine models with high validity at the behavioral level. However, multiple pathogenic mechanisms and complex functional deficits often impose challenges to reliably measure and interpret behavior of chronically sick mice. Therefore, the assessment of peripheral pathology and a behavioral profile at several time points using a battery of tests are required. Video-tracking, behavioral spectroscopy, and remote acquisition of physiological measures are emerging technologies that allow for comprehensive, accurate, and unbiased behavioral analysis in a home-base-like setting. This report describes a refined phenotyping protocol, which includes a custom-made monitoring apparatus (Integrated Behavioral Station, INBEST) that focuses on prolonged measurements of basic functional outputs, such as spontaneous activity, food/water intake and motivated behavior in a relatively stress-free environment. Technical and conceptual improvements in INBEST design may further promote reproducibility and standardization of behavioral studies.

Figure 3 exemplifies different read-outs in a prolonged behavioral study with CD1 mice. Data represents baseline performance (days 6 to 2 prior to surgery), post-surgery recovery (days 2 to 4) and behavioral effects induced by sustained intra-cerebro-ventricular administration of brain-reactive antibodies (days 6 to 10; where 0 denotes the surgery day). Analysis with event-recording software reveals that the experimental group displays impairments in ingestive behavior, as evidenced by a lower frequency of water bottle licks (A), increased latency to approach the sucrose solution (B), and decreased food consumption (C) during the experimental period. Coinciding with these changes, they also show diminished running wheel activity in comparison to control mice (D). As measured by video-tracking software, the experimental group also ambulates less in the home-cage (E) and prefers to spend more time in the shelter (F). These behavioral differences are illustrated on sample ethograms (G).
<img alt="Figure 3A" src="/files/ftp_upload/51524/51524fig3A.jpg" /> 
<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51524/51524fig3Ahighres.jpg" target="_blank">Please click here to view a larger version of this figure.</a> 
<img alt="Figure 3G" src="/files/ftp_upload/51524/51524fig3G.jpg" /> 
<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51524/51524fig3Ghighres.jpg" target="_blank">Please click here to view a larger version of this figure.</a> 
Figure 3. Representative variables in a series of 10-hr daily sessions illustrating the discriminant power of the INBEST system. Experimental mice (exposed to brain-reactive antibodies over 2 weeks) drink less water (A), take longer to approach sucrose solution (B), and consume less food (C) during the testing period. Coinciding with these changes, they also show impaired activity, as exemplified by reduced running wheel count (D), decreased ambulation (E) and prolonged stay in the shelter (F). These behavioral differences are illustrated on sample ethograms (G). The top panel displays the behavior of an experimental mouse on day 6, characterized by reduced ingestive behavior, lower running wheel activity and increased shelter time in comparison to a control mouse receiving a vehicle (bottom panel).

Watch this Scientific Journal Video about Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST at JoVE.com

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST

Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of ...

behavioral-phenotyping-murine-disease-models-with-integrated

Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

9.9K Views.  McMaster University. Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

Video: Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST

Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the &#34;cloud&#34; and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in&#160;cabinet; two such cabinets (16 environments) may be controlled by one computer.

Fully automated system for measuring physiologically meaningful properties of the mechanisms mediating spatial localization, temporal localization, duration, rate&#160;and probability estimation, risk assessment, impulsivity, and the accuracy and precision of memory, in order to assess the effects of genetic and pharmacological manipulations on foundational mechanisms of cognition in mice.

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and ...

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing locomotive impairment in animal models of neuromuscular disease and efficacy of therapeutic drugs that may improve locomotion and/or muscle function. The open field activity measurement provides a different measure than muscle strength, which is commonly assessed by grip strength measurements. It can also show how drugs may affect other body systems as well when used with additional outcome measures. In addition, measures such as total distance traveled mirror the 6 min walk test, a clinical trial outcome measure. However, open field activity monitoring is also associated with significant challenges: Open field activity measurements vary according to animal strain, age, sex, and circadian rhythm. In addition, room temperature, humidity, lighting, noise, and even odor can affect assessment outcomes. Overall, this manuscript provides a well-tested and standardized open field activity SOP for preclinical trials in animal models of neuromuscular diseases. We provide a discussion of important considerations, typical results, data analysis, and detail the strengths and weaknesses of open field testing. In addition, we provide recommendations for optimal study design when using open field activity in a preclinical trial.

Open field activity levels are used to assess locomotive and behavioral activity levels. This protocol provides a well-designed, standardized protocol to use in preclinical trials for neuromuscular disorders.

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer's Disease, Using IntelliCage

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive decline, such as Alzheimer&#39;s disease (AD), have become a promising tool to elucidate the underlying mechanisms and promote the therapeutic advances. An important step is the validation and characterization of expected behavioral abnormality in the models, in the case of AD, cognitive decline. The long-term behavioral investigations of laboratory animals to study the effect of aging demand substantial efforts from researchers. The IntelliCage system is a high-throughput and cost-effective test battery for mice that eliminates the need for daily human handling. Here, we describe how the system is utilized in the long-term phenotyping of a genetic Alzheimer&#39;s disease model, specifically focusing on the cognitive functions. The experiment employs repeated battery of tests that assess spatial learning and executive functions. This cost-effective age-dependent phenotyping allows us to identify the transient and/or permanent effects of genes on various cognitive aspects.

This paper describes a protocol for cognitive assessments for genetic models of the Alzheimer&#39;s disease using the IntelliCage system, which is a high throughput automated behavioral monitoring system with operant conditioning.

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive ...

Behavioral Assessments of Spontaneous Locomotion in a Murine MPTP-induced Parkinson's Disease Model

Parkinson&#8217;s disease (PD) is a common neurodegenerative disorder disease, causing the phenomenon of shaking, rigidity, slowness of movement and dementia. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can lead to some Parkinson&#8217;s-like symptoms by destroying dopaminergic neurons in the substantia nigra of the brain. It has been thus used to establish PD models in various animal studies. Here, mice receive MPTP injections (20 mg/kg/day) for seven days and the behavioral tests are performed on the eighth day. This model is adapted efficiently in the study of PD. The behavioral tests here include the cylinder test and the open field test. The cylinder experiment is used to detect the animals&#8217; ability to lift their front paws when put into a different environment. As the PD model mice show arching&#8212;the mouse arches its back&#8212;the number of paw liftings decrease. This test is easy to execute. The open field test is used to detect the amount of time the mice spend on running, walking, and remaining immobile. We analyze animals&#8217; movements in open field using software and obtain data. Lastly, we use L-DOPA, one of the most commonly used PD drugs, as one example to show how to apply this model to the study of PD drugs. Our results indicate that MPTP neurotoxicity induces motor deficit which can be mitigated by L-DOPA.

We describe establishment of a murine model for Parkinson's disease using MPTP, and behavioral assessments using cylinder and open field tests to measure motor function. We then use L-DOPA as one example to show how to apply this model in the study of PD drugs.

Parkinson&#8217;s disease (PD) is a common neurodegenerative disorder disease, causing the phenomenon of shaking, rigidity, slowness of movement and ...

Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method of gait analysis demonstrated here is a simple but effective measure of gait abnormalities in murine models. Footprints are analyzed by painting a mouse&#8217;s feet with non-toxic washable paint and allowing the subject to walk through a tunnel on a sheet of paper. The design of the testing tunnel takes advantage of natural mouse behavior and their affinity for small dark places. The stride length, stride width, and toe spread of each mouse is easily measured using a ruler and a pencil. This is a well-established and reliable method, and it generates several metrics that are analogous to digital systems. This approach is sensitive enough to detect changes in stride early in phenotype presentation, and due to its non-invasive approach, it allows for testing of groups across life-span or phenotypic presentation.

Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models.

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method ...

A System for Tracking the Dynamics of Social Preference Behavior in Small Rodents

Exploring the neurobiological mechanisms of social behavior requires behavioral tests that can be applied to animal models in an unbiased and observer-independent manner. Since the beginning of the millennium, the three-chamber test has been widely used as a standard paradigm to evaluate sociability (social preference) and social novelty preference in small rodents. However, this test suffers from multiple limitations, including its dependence on spatial navigation and negligence of behavioral dynamics. Presented and validated here is a novel experimental system that offers an alternative to the three-chamber test, while also solving some of its caveats. The system requires a simple and affordable experimental apparatus and publicly available open-source analysis system, which automatically measures and analyzes multiple behavioral parameters at individual and population levels. It allows detailed analysis of the behavioral dynamics of small rodents during any social discrimination test. We demonstrate the efficiency of the system in analyzing the dynamics of social behavior during the social preference and social novelty preference tests as performed by adult male mice and rats. Moreover, we validate the ability of the system to reveal modified dynamics of social behavior in rodents following manipulations such as whisker trimming. Thus, the system allows for rigorous investigation of social behavior and dynamics in small rodent models and supports more accurate comparisons between strains, conditions, and treatments.

Described here is a novel automated experimental system that offers an alternative to the three-chamber test and also solves several caveats. This system supplies multiple behavioral parameters that enable rigorous analysis of small rodent behavioral dynamics during the social preference and social novelty preference tests.

Exploring the neurobiological mechanisms of social behavior requires behavioral tests that can be applied to animal models in an unbiased and ...

Validating a Murine Model of Angelman Syndrome Using Behavioral Tests

Behavioral Characterization of an Angelman Syndrome Mouse Model

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of AS. We use the rotarod learning paradigm, detailed gait analysis, and nest building test to detect and characterize animal motor impairments. We test animal emotionality in the open field and elevated plus maze tests, as well as the affect in the tail suspension test. When AS mice are tested in the open field test, the results should be interpreted with care, since motor dysfunctions influence mouse behavior in the maze and alter activity scores.
The reproducibility and effectiveness of the presented behavioral tests has already been validated in several independent Uba3a mouse lines with different knockout variants, establishing this set of tests as an excellent validation tool in AS research. Models with the relevant construct and face validity will warrant further investigations to elucidate the pathophysiology of the disease and grant the development of causal treatments.

Author Spotlight: A Battery of Highly Reproducible Behavioral Tests to Validate an Angelman Syndrome Murine Model 

This manuscript presents a set of highly reproducible behavioral tests to validate an Angelman syndrome mouse model.

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of ...

Gathering Self-Initiated Rat Behavioral Data to Characterize Post-Stroke Deficits

Behavioral testing in rat models is frequently utilized for diverse purposes, including psychological, biomedical, and behavioral research. Many traditional approaches involve individual, one-on-one testing sessions between a single researcher and each animal in an experiment. This setup can be very time consuming for the researcher, and their presence may impact the behavioral data in unwanted ways. Additionally, traditional caging for rat research imposes a lack of enrichment, exercise, and socialization that would normally be typical for the species, and this context may also skew the results of behavioral data. Overcoming these limitations may be worthwhile for several research applications, including the study of acquired brain injury. Here, an example method is presented for automatically training and testing individual rat behavior in a colony cage without the presence of humans. Radio frequency identification can be utilized to tailor sessions to the individual rat. The validation of this system occurred in the example context of measuring skilled forelimb motor performance before and after stroke. Traditional characteristics of post-stroke behavioral impairments and novel measures enabled by the system are measured, including success rate, various aspects of pull force, bout analysis, initiation rate and patterns, session duration, and circadian patterns. These variables can be collected automatically with few limitations; though the apparatus removes experimental control of exposure, timing and practice, the validation produced reasonable consistency in these variables from animal to animal.

A system for acquiring data from self-initiated individual behavior sessions within a social colony cage setting is presented. The efficacy of this system is demonstrated using an automated skilled reach assessment, enabling the characterization of post-stroke motor impairments, potential behavioral alterations related to motivation, circadian variations, and other innovative dependent variables.

Behavioral testing in rat models is frequently utilized for diverse purposes, including psychological, biomedical, and behavioral research. Many ...

Behavioral Assays for Autism Spectrum Disorder in Mouse Models

Strategies for Assessing Autistic-Like Behaviors in Mice

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social communication impairments and restrictive or repetitive behaviors, such as hand flapping or lining up objects. These behavioral patterns can be reliably observed in mouse models with ASD-linked genetic mutations, making them highly useful tools for studying the underlying cellular and molecular mechanisms in ASD. Understanding how genetic changes affect the neurobiology and behaviors observed in ASD will facilitate the development of novel targeted therapeutic compounds to ameliorate core behavioral impairments. Our lab has employed several protocols encompassing well-described training and testing procedures that reflect a wide range of behavioral deficits related to ASD. Here, we detail two assays to study the core features of ASD in mouse models: self-grooming (a measure of repetitive behavior) and the three-chamber social interaction test (a measure of social interaction approach and preference for social novelty).

Rodent models are valuable tools for studying core behaviors related to autism spectrum disorder (ASD). In this article, we expound on two behavioral tests for modeling the core features of ASD in mice: self-grooming, which assesses repetitive behavior, and the three-chamber social interaction test, which documents social impairments.

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social ...