Name: behavioral phenotyping of murine disease models with the integrated behavioral station (inbest)
Uploaded: 2015-04-23T13:00:00
Duration: 12 min 18 s
Description: Watch this Scientific Journal Video about Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST at JoVE.com

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://www.jove.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 border="0" cellpadding="0" cellspacing="0">
	<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 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://www.jove.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://www.jove.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

Research

JoVE Journal

Behavior

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

The minimization and alleviation of  suffering has moral and scientific implications. In order to mitigate this  negative experience one must be able to ...

Behavioral Assessment of the Aging Mouse Vestibular System

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of studies show changes in balance phenotype with age in rodents, very few isolate the vestibular contribution to balance under either normal conditions or during senescence. We use two standard behavioral tests to characterize the balance performance of mice at defined age points over the lifespan: the rotarod test and the inclined balance beam test. Importantly though, a custom built rotator is also used to stimulate the vestibular system of mice (without inducing overt signs of motion sickness). These two tests have been used to show that changes in vestibular mediated-balance performance are present over the murine lifespan. Preliminary results show that both the rotarod test and the modified balance beam test can be used to identify changes in balance performance during aging as an alternative to more difficult and invasive techniques such as vestibulo-ocular (VOR) measurements.

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of ...

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2.
In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells.
The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

Performing Behavioral Tasks in Subjects with Intracranial Electrodes

Patients having stereo-electroencephalography (SEEG) electrode, subdural grid or depth electrode implants have a multitude of electrodes implanted in different areas of their brain for the localization of their seizure focus and eloquent areas. After implantation, the patient must remain in the hospital until the pathological area of brain is found and possibly resected. During this time, these patients offer a unique opportunity to the research community because any number of behavioral paradigms can be performed to uncover the neural correlates that guide behavior. Here we present a method for recording brain activity from intracranial implants as subjects perform a behavioral task designed to assess decision-making and reward encoding. All electrophysiological data from the intracranial electrodes are recorded during the behavioral task, allowing for the examination of the many brain areas involved in a single function at time scales relevant to behavior. Moreover, and unlike animal studies, human patients can learn a wide variety of behavioral tasks quickly, allowing for the ability to perform more than one task in the same subject or for performing controls. Despite the many advantages of this technique for understanding human brain function, there are also methodological limitations that we discuss, including environmental factors, analgesic effects, time constraints and recordings from diseased tissue. This method may be easily implemented by any institution that performs intracranial assessments; providing the opportunity to directly examine human brain function during behavior.

Patients implanted with intracranial electrodes provide a unique opportunity to record neurological data from multiple areas of the brain while the patient performs behavioral tasks. Here, we present a method of recording from implanted patients that can be reproducible at other institutions with access to this patient population.

Patients having stereo-electroencephalography (SEEG) electrode, subdural grid or depth electrode implants have a multitude of electrodes implanted in ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a series of basic techniques are described to enable observations of the reproductive behavior of a wild-caught gobiid fish, as a model, kept in an aquarium. The method focuses on three steps: collection, transport, and observations of reproductive ecology of a substrate spawner. Essential aspects of live fish collection and transport are (1) preventing injury to the fish, and (2) careful acclimation to the aquarium. Preventing harm through injuries such as scratches or a sudden change of water pressure is imperative when collecting live fish, as any physical damage is likely to negatively affect the survival and later behavior of the fish. Careful acclimation to aquaria decreases the incidence death and mitigates the shock of transport. Observations during captive rearing include (1) the identification of individual fish and (2) monitoring spawned eggs without negative effects to the fish or eggs, thereby enabling detailed investigation of the study species' reproductive ecology. The subcutaneous injection of a visible implant elastomer (VIE) tag is a precise method for the subsequent identification of individual fish, and it can be used with a wide size range of fish, with minimal influence on their survival and behavior. If the study species is a substrate spawner that deposits adhesive eggs, an artificial nest site constructed from polyvinyl chloride (PVC) pipe with the addition of a removable waterproof sheet will facilitate counting and monitoring the eggs, lessening the investigator's influence on the nest-holding and egg-guarding behavior of the fish. Although this basic method entails techniques that are seldom mentioned in detail in research articles, they are fundamental for undertaking experiments that require the captive rearing of a wild fish.

A series of basic methods to enable the study of the reproductive ecology of fish kept in aquaria are described. These are useful protocols for collecting fish using SCUBA, transporting live fish, and observing the reproductive behavior of wild-caught fish kept in aquaria.

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a ...

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.

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer&#039;s Disease, Using IntelliCage

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 ...

The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by many patients prompts the need to develop new therapeutic alternatives and to better understand the etiology of the disorder. Pre-clinical models with translational merits are rudimentary for this task. Here we present a protocol for the unpredictable chronic mild stress (UCMS) method in mice. In this protocol, adolescent mice are chronically exposed to interchanging unpredictable mild stressors. Resembling the pathogenesis of depression in humans, stress exposure during the sensitive period of mice adolescence instigates a depressive-like phenotype evident in adulthood. UCMS can be used for screenings of antidepressants on the variety of depressive-like behaviors and neuromolecular indices. Among the more prominent tests to assess depressive-like behavior in rodents is the sucrose preference test (SPT), which reflects anhedonia (core symptom of depression). The SPT will also be presented in this protocol. The ability of UCMS to induce anhedonia, instigate long-term behavioral deficits and enable reversal of these deficits via chronic (but not acute) treatment with antidepressants strengthens the protocol&#39;s validity compared to other animal protocols for inducing depressive-like behaviors.

Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by ...

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.

Behavioral Assessments of Spontaneous Locomotion in a Murine MPTP-induced Parkinson&#039;s Disease Model

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 ...