This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) as follows: 15H05724, 16H01491, and 15H04289.

All experiments were performed in accordance with the guidelines for animal experiments from the National Institute of Genetics (NIG), and all procedures were approved by the NIG Committee for Animal Care and Use (approval no. 26-9).
1. Animals
<ol>
	<li>Selective breeding
	<ol>
		<li>Create wild-derived heterogeneous stock (WHS)6 mice by crossing 8 inbred strains (MSM, HMI, BLG2, PGN2, KJR, CHD, NJL, and BFM/2) while avoiding any inbreeding (mating between siblings or cousins)7. Conduct a selective breeding, starting in the third generation (G3), by selecting mice that display longer &#8220;contact time&#8221; (selected group, 16 families of mating pairs) according to the active tameness test (see step 3.1).</li>
		<li>Generate the non-selected control group (also 16 families of mating pairs) via random mating from G3 onward. For a representative experiment, use mice obtained from selected (S1) and non-selected (C1) groups of WHS mice to show the pattern of both active and passive tameness using the three tameness tests (see step 3.1 - 3.3). For the experiments, use 10 G15 mice per group (S1 and C1).</li>
	</ol>
	</li>
	<li>Representative subject animals
	<ol>
		<li>Wean all mice from their parents at 3 weeks of age and house them in same-sex groups with littermates, using standard-size plastic cages containing wood chips, until the test. Maintain all animals at an NIG animal facility under a 12/12 h light/dark cycle (lights on from 06:00 to 18:00) and a constant temperature (23 &#177; 2 &#176;C).</li>
		<li>Ensure that food and water are available ad libitum. Test the mice at 6 weeks of age during the light period to analyze their interactions with humans during the part of the day when humans are typically active.</li>
	</ol>
	</li>
	<li>Handling protocol
	<ol>
		<li>Use large tweezers covered with silicon tubes to transfer the mice to new cages by picking them up gently by their tails. Use the tweezers to separate them from their parents at weaning and move them during the experiments. None of the mice should receive handling before the tameness tests.</li>
		<li>Transfer the mice to an experimental room just before the tameness tests.</li>
		<li>Keep the male mice separated from one another after the test, to avoid inter-male aggression in WHS mice.</li>
	</ol>
	</li>
</ol>
2. Equipment
<ol>
	<li>Use an open-field apparatus (400 x 400 x 400 mm, grayish polyvinylchloride).</li>
	<li>Illuminate the open field using a clip light with a neutral white bulb (100 lux) fixed at a height of 450 mm above the open field (Figure 1).</li>
	<li>Record all behaviors using a commercially available compact digital camera fixed at 850 mm above the open field using a tripod (Figure 1).</li>
	<li>Wear cloth gloves under latex gloves to protect the hands from being bitten at during the passive tameness test (a rare reaction).</li>
	<li>Measure the test period using a stopwatch.</li>
</ol>
3. Tests
Note: The first two tests (active tameness and passive tameness) were conducted for 1 min each, and the stay-on-hand test was conducted for less than 1 min. All three tests were conducted in the open-field apparatus and recorded using a digital camera.&#160;Iodophore disinfection procedure should be performed on all apparats before any behavior studies.
<ol>
	<li>Procedures for active tameness test
	<ol>
		<li>Turn on the digital camera and start recording.</li>
		<li>Pick up a mouse gently by the tail using a pair of large tweezers (see step 1.3.1) and transfer it from the home cage to the center of the open-field apparatus.</li>
		<li>Start the stopwatch.</li>
		<li>Place the left hand on the lower left corner of the apparatus.</li>
		<li>Keep the palm facing up and move the fingers slowly and steadily to ensure that the mouse connects the hand to a human. The average time to move all four fingers from the little finger to the index finger should be approximately 0.5 s, with a continuously repeated movement cycle.</li>
		<li>Move the hand toward the mouse slowly, at an average speed of about 13 cm/s, stopping at 10 cm from the side of the mouse&#8217;s body.</li>
		<li>Maintain the same distance if the mouse attempts to move away. If the mouse approaches, keep the hand in the same position and continue to move the fingers slightly. If the mouse attempts to climb up the arm, return it to the field using the free hand.</li>
		<li>Conclude the test after 1 min, withdraw the hand, and proceed to the passive tameness test.</li>
	</ol>
	</li>
	<li>Procedures for passive tameness test
	<ol>
		<li>Follow the same steps as for the active tameness test, but instead of stopping 10 cm away from the mouse&#8217;s body, slowly touch the mouse with the fingertips.</li>
		<li>Leave the fingers in the same position, contacting the mouse, until the mouse moves away. When the mouse attempts to break contact, follow it, moving at an average speed of about 23 cm/s, and reinitiate the touch.</li>
		<li>Conclude the test after 1 min. Restart the stopwatch immediately to begin the stay-on-hand test.</li>
	</ol>
	</li>
	<li>Procedures for stay-on-hand test
	<ol>
		<li>Lift the mouse by the tail using a pair of large tweezers and place it gently on the left palm. Keep the hand close to the floor of the open-field apparatus.</li>
		<li>Stroke the mouse gently at an average speed of 0.5 s/stroke using the left thumb until it leaves the hand.</li>
		<li>Repeat steps 3.3.1 - 3.3.2 3x.</li>
		<li>After the third trial, stop the video recording; all tameness tests are now concluded.</li>
	</ol>
	</li>
	<li>Post-experimental procedures
	<ol>
		<li>Weigh the mouse and count the number of feces and urine left in the open field.</li>
		<li>Mark the mouse individually via an ear punch.</li>
		<li>Wipe the apparatus and experimenter gloves with a paper towel soaked in 0.2% Iodophor sanitizer.</li>
		<li>Clean the apparatus with a dry paper towel.</li>
	</ol>
	</li>
</ol>
4. Behavioral analyses
Note: Video recordings were scored using tanaMove version 0.01 (Windows 7), event-recording software for behavioral analyses (Figure 2). The software is freely available (Supplemental Materials 1) and from our website (http://www.mgrl-lab.jp/MGRL_software.html). An individual who was blind to the test conditions scored all footage. The duration of all behavioral indices was measured in 0.1-s increments. During the scoring, the observer pressed the appropriate letter on the keyboard (see step 4.1) upon noting a given behavior.
<ol>
	<li>Assignment of keys for video analysis
	<ol>
		<li>Assign keys for each behavior index. Using appropriate keys for each behavioral index is critical to summarize the data automatically in step 4.3. 
		Note: The assignments used in this study are shown in Figure 3.</li>
	</ol>
	</li>
	<li>Video analysis
	<ol>
		<li>Analyze each video recording 3x for different behavioral indices (Figure 3B). Self-grooming behavior was not scored. Make sure the built-in speaker of the computer is switched on in order to hear the timer sound in the video.</li>
		<li>Launch tanaMove.</li>
		<li>Drag and drop the video file to be analyzed into the window of tanaMove. Confirm that the file is loaded and that the path and name of the outputted CSV file in the Data File [.csv] box is correct.</li>
		<li>Click the Ready button. This activates a new window to enter the assigned keys.</li>
		<li>Click the Play button to start the video file. Click the Stop button to pause the video file.</li>
		<li>Press key A when the mouse is observed &#8220;heading&#8221; toward or approaching the hand, including standing to sniff. Press key R when the mouse is observed &#8220;locomoting&#8221; without heading toward the hand during the active tameness test.</li>
		<li>Press key W for at least 0.3 s (at least three intervals in the window) when the timer sound is heard at 60 s.</li>
		<li>Initiate the analysis of the passive tameness test after the left hand enters the video. Press key A when the mouse is observed heading toward or approaching the hand and key R when the mouse is observed traveling without heading toward the hand during the passive tameness test.</li>
		<li>Press key W for at least 0.3 s (at least three intervals in the window) when the timer sound is heard at 60 s.</li>
		<li>Press key T when the mouse is observed &#8220;staying&#8221; on the hand in the stay-on-hand test.</li>
		<li>After the stay-on-hand test, click the Stop button to stop the video observation.</li>
		<li>Click the Back button and type a new file name for the CSV data output.</li>
		<li>Click the Ready button to activate a new window to enter the assigned keys.</li>
		<li>Click the Play button to restart the video footage and initiate the observation of the active tameness test.</li>
		<li>Press key T when the mouse is observed &#8220;contacting&#8221; the human, which includes all voluntary contacting behavior but not accidental contacts, such as landing on a palm after jumping, during the active tameness test.</li>
		<li>Press key W for at least 0.3 s (at least three intervals in the window) when the timer sound is heard at 60 s.</li>
		<li>Press key T when the mouse allows the experimenter to touch its body during the passive tameness test.</li>
		<li>Press key W for at least 0.3 s (at least three intervals in the window) when the timer sound is heard at 60 s.</li>
		<li>Click the Stop button to stop the video.</li>
		<li>Click the Back button and type a new file name for the CSV data output.</li>
		<li>Click the Ready button to activate a new window to enter the assigned keys.</li>
		<li>Click the Play button to restart the video footage and initiate the observation of the active tameness test.</li>
		<li>Press key J when the mouse is observed &#8220;jumping&#8221; during the active tameness test.</li>
		<li>Press key W for at least 0.3 s (at least three intervals in the window) when the timer sound is heard at 60 s. Then start the observation of the passive tameness test.</li>
		<li>Press key J when the mouse is observed &#8220;jumping&#8221; during the passive tameness test.</li>
		<li>Click the Stop button to stop the video observation when the timer sound is heard at 60 s.</li>
		<li>Click the Back button and type a new file name for the CSV data output.</li>
	</ol>
	</li>
	<li>Summarize the data
	<ol>
		<li>Save all the raw data files (CSV) in the same folder with the Supplementary 2.xlsm file (Supplemental Materials 2).</li>
		<li>Open the Supplementary 2.xlsm file and click the Summarize button.</li>
		<li>Save the results as a separate file and use it for further analysis of the tameness behavior.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Albert, F. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+differences+in+behavior,+physiology+and+neurochemistry+between+rats+selected+for+tameness+and+for+defensive+aggression+towards+humans.">Phenotypic differences in behavior, physiology and neurochemistry between rats selected for tameness and for defensive aggression towards humans.</a> Hormones and Behavior. 53 (3), 413-421 (2008).</li><li>Belyaev, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Destabilizing+selection+as+a+factor+in+domestication.">Destabilizing selection as a factor in domestication.</a> Journal of Heredity. 70 (5), 301-308 (1978).</li><li>Trut, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+canid+domestication:+the+farm-fox+experiment.">Early canid domestication: the farm-fox experiment.</a> American Scientist. 87 (2), 160-169 (1999).</li><li>Price, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Animal Domestication and Behavior. , (2002).</li><li>Goto, T., Tanave, A., Moriwaki, K., Shiroishi, T., Koide, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+for+reluctance+to+avoid+humans+during+the+domestication+of+mice.">Selection for reluctance to avoid humans during the domestication of mice.</a> Genes, Brain and Behavior. 12 (8), 760-770 (2013).</li><li>Matsumoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+breeding+and+selection+mapping+using+a+novel+wild-derived+heterogeneous+stock+of+mice+revealed+two+closely+linked+loci+for+tameness.">Selective breeding and selection mapping using a novel wild-derived heterogeneous stock of mice revealed two closely linked loci for tameness.</a> Scientific Reports. 7, 4607 (2017).</li><li>Koide, T., Goto, T., Takano-Shimizu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+mixing+to+elucidate+the+genetic+system+of+complex+traits.">Genomic mixing to elucidate the genetic system of complex traits.</a> Experimental Animals. 61 (5), 503-509 (2012).</li><li>Albert, F. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+architecture+of+tameness+in+a+rat+model+of+animal+domestication.">Genetic architecture of tameness in a rat model of animal domestication.</a> Genetics. 182 (2), 541-554 (2009).</li><li>Cottle, C. A., Price, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+nonagouti+pelage-color+allele+on+the+behavior+of+captive+wild+Norway+rats+(Rattus+norvegicus).">Effects of the nonagouti pelage-color allele on the behavior of captive wild Norway rats (Rattus norvegicus).</a> Journal of Comparative Psychology. 101 (4), 390-394 (1987).</li><li>Hayssen, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+nonagouti+coat-color+allele+on+behavior+of+deer+mice+(Peromyscus+maniculatus):+a+comparison+with+Norway+rats+(Rattus+norvegicus).">Effects of the nonagouti coat-color allele on behavior of deer mice (Peromyscus maniculatus): a comparison with Norway rats (Rattus norvegicus).</a> Journal of Comparative Psychology. 111 (4), 419-423 (1997).</li><li>Arakawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+male-specific+QTL+for+social+interaction+behavior+in+mice+mapped+with+automated+pattern+detection+by+a+hidden+Markov+model+incorporated+into+newly+developed+freeware.">A male-specific QTL for social interaction behavior in mice mapped with automated pattern detection by a hidden Markov model incorporated into newly developed freeware.</a> Journal of Neuroscience Methods. 234, 127-134 (2014).</li></ol>

The authors have nothing to disclose.

The representative results cover nine behavioral indices from the active tameness, passive tameness, and stay-on-hand tests. A comparison of the scores between the selected and control groups revealed clear differences in tameness. These results show that the three tests are valid for measuring tameness in mice.
Tameness levels in animals can be difficult to quantify empirically. Several methods have been developed for evaluating tameness in deer, mice, and rats8,<...

Selective breeding during domestication has changed the characteristics of domesticated animals to make them better adapted for living with humans1,2,3. &#34;Tameness&#34; is a common behavioral trait observed in domesticated animals. The studies of selective breeding for tameness in animals reveal how their phenotypes have changed in the course of domestication. In a famous study, foxes were selectively bred for tameness and a group of foxes that exhibited a high level of tameness was established2. The foxes also showed other phenotype changes involving morphology, reproduction, coat color, and physiological traits. Selective breeding for tameness in mice, a popular laboratory animal, can help reveal the mechanism underlying the change in a variety of phenotypes during domestication.
Tameness has two behavioral components: active approaches to humans (active tameness) and a reduction in the avoidance of humans (passive tameness)4. In a previous study, we established three behavioral tests to characterize the tameness levels in mice: active tameness, passive tameness, and stay-on-hand tests5. The first test was designed to quantify a mouse&#39;s active tameness by measuring the latency of its spontaneous approach toward a human and the time it spent in contact with a human hand. The second test was designed to quantify passive tameness by measuring how long a mouse tolerated being touched by a human hand. The stay-on-hand test was intended to quantify behavioral avoidance of being stroked mildly by a human hand. These three assessments require only an open-field apparatus, a digital camera, and a large pair of tweezers. They can be completed sequentially within 3 min, enabling a large quantity of data to be collected per day.
In the previous study, we characterized and compared tameness behaviors using a series of inbred mouse strains: 10 wild strains, one Japanese fancy mouse-derived strain, and six laboratory strains5. Most domesticated strains showed a significantly higher passive tameness than wild strains, but the active tameness did not differ between the two groups. From these results, we concluded that the domesticated mice that are the founders of laboratory strains were predominantly selected for their passive tameness, but not for active tameness, in their breeding history.
We next conducted selective breeding with eight wild mouse strains to generate a genetically heterogeneous population6. We chose the animals that scored high on the active tameness test and successfully increased their active tameness in successive generations, compared with a control group. This outcome indicated that the tests reliably measure the tameness in mice.
Here, we describe the comprehensive method for measuring behavioral indices using the three behavioral tests. First, we explain the set-up and necessary apparatus. Next, we detail the step-by-step procedure for running the experiments, collecting data through video, and analyzing said data using freeware that we developed. Finally, we show representative results comparing the pool of selectively bred mice with a control group.

<table><tbody><tr><td>open-field apparatus</td><td>O&amp;rsquo;Hara &amp;amp; Co. Ltd.</td><td>OF-3102</td><td>400&amp;times;400&amp;times;400 mm, grayish polyvinylchloride</td></tr><tr><td>compact digital camera</td><td>Ricoh Company, Ltd.</td><td>CX-5</td><td>production in the company is over</td></tr><tr><td>latex pure gloves</td><td>AS ONE</td><td>BioLab, IKG-8002</td><td></td></tr><tr><td>stopwatch</td><td>SEIKO</td><td>SSBJ018</td><td></td></tr><tr><td>Iodophor sanitizer</td><td>Ecolab G.K. (CLEA Japan)</td><td>CL-4123</td><td>Mikro Klene</td></tr></tbody></table>

measuring active and passive tameness separately in mice

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

We previously conducted selective breeding for active tameness in miceandsuccessfully produced mice that showed a high level of active tameness6. The representative results of the tameness tests for the selected and non-selected groups of mice at the 15th generation (G15) are shown in Figure 4. Mice from the selected group scored higher on the active tameness test, exhibiting a significantly longer duration of contact with a...

Watch this Scientific Journal Video about Measuring Active and Passive Tameness Separately in Mice at JoVE.com

Measuring Active and Passive Tameness Separately in Mice

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

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

measuring-active-and-passive-tameness-separately-in-mice

Research

JoVE Journal

Behavior

7.1K Views.  National Institute of Genetics. Tameness bij dieren omvat een verlaging van vermijden reacties bij de mens (passieve tameness) en een toename in actieve benaderingen voor de mens (actieve tameness). Hier beschrijven we gedetailleerde protocollen voor drie gedrags proeven (actieve tameness, passieve tameness en verblijf-over-hand testen) apart meten de actieve en passieve tameness van muizen.

Video: Measuring Active and Passive Tameness Separately in Mice

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.

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.

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

Measuring the Strength of Mice

Kondziela7 devised the inverted screen test and published it in 1964. It is a test of muscle strength using all four limbs. Most normal mice easily score maximum on this task; it is a quick but insensitive gross screen, and the weights test described in this article will provide a finer measure of muscular strength.
There are also several strain gauge-based pieces of apparatus available commercially that will provide more graded data than the inverted screen test, but their cost may put them beyond the reach of many laboratories which do not specialize in strength testing. Hence in 2000 a cheap and simple apparatus was devised by the author. It consists of a series of chain links of increasing length, attached to a "fur collector" a ball of fine wire mesh sold for preventing limescale build up in hard water areas. An accidental observation revealed that mice could grip these very tightly, so they proved ideal as a grip point for a weight-lifting apparatus. A common fault with commercial strength meters is that the bar or other grip feature is not thin enough for mice to exert a maximum grip. As a general rule, the thinner the wire or bar, the better a mouse can grip with its small claws.
This is a pure test of strength, although as for any test motivational factors could potentially play a role. The use of scale collectors, however, seems to minimize motivational problems as the motivation appears to be very high for most normal young adult mice.

Deficits in muscular strength occur in many clinical conditions such as motor neuron disease. The inverted screen and weight lifting tests described here measure strength in mice almost exclusively, with minimal influence of factors such as coordination.

Kondziela7 devised the inverted screen test and published it in 1964. It is a test of muscle strength using all four limbs. Most normal mice easily score ...

Medicine

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of ...

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the test mouse is placed in a standardized apparatus and relevant behaviors are assessed in three different sessions (called session I, II, and III).
The apparatus has three compartments (see Figure 1), the left and right compartments contain an inverted cup which can house a mouse (called &#34;stimulus mouse&#34;).
In session I, the test mouse is placed in the cage and its mobility is characterized by the number of transitions made between compartments. In session II, a stimulus mouse is placed under one of the inverted cups and the sociability of the test mouse is quantified by the amounts of time it spends near the cup containing the enclosed stimulus mouse vs. the empty inverted cup. In session III, the inverted cups are removed and both mice interact freely. The sociability of the test mouse in session III is quantified by the number of social approaches it makes toward the stimulus mouse and by the number of times it avoids a social approach by the stimulus mouse.
The automated evaluation of the movie detects the nose of the test mouse, which allows the determination of all described sociability measures in session I and II (in session III, approaches are identified automatically but classified manually). To find the nose, the image of an empty cage is digitally subtracted from each frame of the movie and the resulting image is binarized to identify the mouse pixels. The mouse tail is automatically removed and the two most distant points of the remaining mouse are determined; these are close to nose and base of tail. By analyzing the motion of the mouse and using continuity arguments, the nose is identified.
<img alt="Figure 1" src="/files/ftp_upload/52508/52508fig1.jpg" /> 
Figure 1. Assessment of Sociability During 3 sessions. Session I (top): Acclimation of test mouse to the cage. Session II (middle): Test mouse moving freely in the cage while the stimulus mouse is enclosed in an inverted cup. Session III (bottom): Both test mouse and stimulus mouse are allowed to move freely and interact with each other.

This protocol describes a method to quantify mouse sociability. Mice are videotaped as they move and interact in a special cage. Movie processing allows for the automated quantification of sociability with excellent accuracy and reliability.

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Neuroscience

The Tail Suspension Test

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that are expected to affect depression related behaviors. Mice are suspended by their tails with tape, in such a position that it cannot escape or hold on to nearby surfaces. During this test, typically six minutes in duration, the resulting escape oriented behaviors are quantified. The tail-suspension test is a valuable tool in drug discovery for high-throughput screening of prospective antidepressant compounds. Here, we describe the details required for implementation of this test with additional emphasis on potential problems that may occur and how to avoid them. We also offer a solution to the tail climbing behavior, a common problem that renders this test useless in some mouse strains, such as the widely used C57BL/6. Specifically, we prevent tail climbing behaviors by passing mouse tails through a small plastic cylinder prior to suspension. Finally, we detail how to manually score the behaviors that are manifested in this test.

The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that ...

Assessment of Sexual Behavior of Male Mice

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral pattern. The purpose of this article is to describe the hormone-induced estrus ovariectomized female model and the experimental procedure for the assessment of sexual behavior of male mice. The most important sexual behavioral elements are demonstrated in the video and illustrations. The critical steps, advantages, and limitations of the sexual behavior test are explained as well. Finally, the behavior parameters are presented, and mounting, intromission, and ejaculation processes in mating are distinguished. Behavioral parameters are assessed in terms of the occurred duration and counts during the test period.

This article describes how to perform sexual behavior tests in male mice.

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral ...

Studying Active Defensive Behaviors in Mice with Modified Fear Conditioning

Modified Fear Conditioning for Inducing Flight Behaviors in Mice

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active defensive behavior, such as jumping or rapid darting, is expressed under high threat imminence or actual threat, whereas passive defensive behavior, such as freezing, is expressed when the threat is predicted, but the threat imminence is relatively low. In classical fear conditioning, subjects typically exhibit freezing as a conditioned defensive response, with little expression of active defensive behavior in most cases. Here, we introduce a modified fear conditioning procedure for mice to observe the transition from freezing to flight and vice versa, involving five repetitive pairings of conditioned stimuli (CS; continuous tone, 8 kHz, 95 dB SPL (sound pressure levels)) and unconditioned stimuli (US; foot shock, 0.9 mA, 1.0 s) over two days. This modified fear conditioning procedure requires a relatively large number of conditioning sessions and conditioning days but does not necessitate a high-intensity foot shock for modest expression of flight behavior. Using the same context for conditioning and salient CS presentations is essential to elicit flight behaviors. This modified fear conditioning procedure is a reliable method for observing active defensive behaviors in mice, providing an opportunity to elucidate the fine mechanisms and characteristics of such behaviors in a fearful context.

Author Spotlight: Exploring Neural Correlates of Defensive Behaviors in Fear Learning and Extinction

To study flight behaviors in a fearful context, we introduce a modified fear conditioning protocol. This protocol ensures that mice consistently exhibit flight behaviors during cue presentation in the fear conditioning.

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active ...

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 ...

Tracking Individual Running Metrics in Mice Using a Voluntary Wheel Running Protocol that Minimizes Social Isolation

Voluntary wheel running (VWR) in rodents is used to evaluate performance and endurance and to simulate exercise. In contrast to other exercise paradigms that require aversive stimulus to induce active movement, wheel running is voluntary. VWR generally consists of placing a running wheel inside the mouse home cage. However, analyzing exercise parameters can typically only be conducted when mice are individually housed, while overall cage effects can be reported when mice are group-housed. Therefore, the current models force experimenters to choose between measuring individual exercise parameters in single-housed mice or measuring group effects in group-housed mice; depending on the research question and model, this could be limiting. 
Therefore, the goal of this method is to demonstrate a protocol that assesses VWR in a mouse model by quantifying running parameters in individual mice, with minimal isolation. We report on device design, preoperative preparations, and experiment setup. Results of a 6-week training protocol using this VWR model suggest it provides sufficient stimulus to increase spatial object recognition memory, comparable to the clinical effect of exercise. This simple and inexpensive model facilitates investigation into the effect of VWR on multiple measurements of wheel activity, minimizing factors that could limit or affect experimental outcomes.

This protocol describes an effective voluntary wheel-running model that can be implemented in various research contexts. Unlike other voluntary running models, this method avoids isolation housing while tracking running metrics in individual mice.

Voluntary wheel running (VWR) in rodents is used to evaluate performance and endurance and to simulate exercise. In contrast to other exercise paradigms ...

Measuring Active and Passive Tameness Separately in Mice

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