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 border="0" cellpadding="0" cellspacing="0" width="818">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">open-field apparatus</td>
			<td style="width:169px;">O&#8217;Hara &#38; Co. Ltd.</td>
			<td style="width:151px;">OF-3102</td>
			<td style="width:283px;">400&#215;400&#215;400 mm, grayish polyvinylchloride</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">compact digital camera</td>
			<td>Ricoh Company, Ltd.</td>
			<td>CX-5</td>
			<td>production in the company is over</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">latex pure gloves</td>
			<td>AS ONE</td>
			<td>BioLab, IKG-8002</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">stopwatch</td>
			<td>SEIKO</td>
			<td>SSBJ018</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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

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Behavior

7.0K Views.  National Institute of Genetics. 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.

Video: Measuring Active and Passive Tameness Separately in Mice

The Successive Alleys Test of Anxiety in Mice and Rats

The plus-maze was derived from the early work of Montgomery. He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones. Handley, Mithani and File et al. performed the first studies on the plus-maze design we use today, and in 1987 Lister published a design for use with mice. 
Time spent on, and entries into, the open arms are an index of anxiety; the lower these indices, the more anxious the mouse is. Alternatively, a mouse that spends most of its time in the closed arms is classed as anxious. 
One of the problems of the plus-maze is that, while time spent on, and entries into, the open arms is a fairly unambiguous measure of anxiety, time in the central area is more difficult to interpret, although time spent here has been classified as &#x201C;decision making&#x201D;. In many tests central area time is a considerable part of the total test time.
Shepherd et al. produced an ingenious design to eliminate the central area, which they called the &#x201C;zero maze&#x201D;. However, although used by several groups, it has never been as widely adopted as the plus-maze. 
In the present article I describe a modification of the plus-maze design that not only eliminates the central area but also incorporates elements from other anxiety tests, such as the light-dark box and emergence tests. It is a linear series of four alleys, each having increasing anxiogenic properties. It has given similar results to the plus-maze in general. Although it may not be more sensitive than the plus-maze (more data is needed before a firm conclusion can be reached on this point), it provides a useful confirmation of plus-maze results which would be useful when, for example, only a single example of a mutant mouse was available, as, for example, in ENU-based mutagenesis programs.

The plus-maze measures anxiety-like behaviour in rodents. There are two opposite closed and two opposite open arms; anxious rodents avoid the open arms. The central area is neither completely open nor closed, so time spent here is ambiguous and difficult to interpret. Here a modification of the plus-maze protocol eliminating this area is described.

The plus-maze was derived from the early work of Montgomery. He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones.  ...

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.

In the following experiment we describe a protocol for trace fear conditioning in mice. This type of associative memory includes a trace period that separates the neutral stimulus and the unconditioned stimulus.

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings ...

Measuring Attentional Biases for Threat in Children and Adults

Investigators have long been interested in the human propensity for the rapid detection of threatening stimuli. However, until recently, research in this domain has focused almost exclusively on adult participants, completely ignoring the topic of threat detection over the course of development. One of the biggest reasons for the lack of developmental work in this area is likely the absence of a reliable paradigm that can measure perceptual biases for threat in children. To address this issue, we recently designed a modified visual search paradigm similar to the standard adult paradigm that is appropriate for studying threat detection in preschool-aged participants. Here we describe this new procedure. In the general paradigm, we present participants with matrices of color photographs, and ask them to find and touch a target on the screen. Latency to touch the target is recorded. Using a touch-screen monitor makes the procedure simple and easy, allowing us to collect data in participants ranging from 3 years of age to adults. Thus far, the paradigm has consistently shown that both adults and children detect threatening stimuli (e.g., snakes, spiders, angry/fearful faces) more quickly than neutral stimuli (e.g., flowers, mushrooms, happy/neutral faces). Altogether, this procedure provides an important new tool for researchers interested in studying the development of attentional biases for threat.

Here we describe a touch-screen visual search paradigm that can be used to study threat detection across the lifespan. The paradigm has already been used in various studies demonstrating that both children and adults detect threatening stimuli like snakes, spiders, and angry faces faster than non-threatening stimuli.

Investigators have long been interested in the human propensity for the rapid detection of threatening stimuli. However, until recently, research in this ...

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 ...

Testing for Odor Discrimination and Habituation in Mice

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the mouse can discriminate between non-social odors and social odors, whether the mouse habituates to a repeatedly presented odor, and whether the mouse demonstrates dishabituation when presented with a novel odor. Since many social behavior tests measure the experimental animal&#8217;s response to a familiar or novel mouse, false positives can be avoided by establishing that the animals can detect and discriminate between social odors. There are similar considerations in learning tests such as fear conditioning that use odor to create a novel environment or olfactory cues as an associative stimulus. Deficits in the olfactory system would impair the ability to distinguish between contexts and to form an association with an olfactory cue during fear conditioning. 
In the odor habitation/dishabituation test, the mouse is repeatedly presented with several odors. Each odor is presented three times for two minutes. The investigator records the sniffing time directed towards the odor as the measurement of olfactory responsiveness. A typical mouse shows a decrease in response to the odor over repeated presentations (habituation). The experimenter then presents a novel odor that elicits increased sniffing towards the new odor (dishabituation). After repeated presentation of the novel odor the animal again shows habituation. This protocol involves the presentation of water, two or more non-social odors, and two social odors. In addition to reducing experimental confounds, this test can provide information on the function of the olfactory systems of new knockout, knock-in, and conditional knockout mouse lines.

This manuscript describes a protocol to examine the olfactory system of rodents. The olfactory habituation/dishabituation test will allow investigators to determine whether a mouse habituates to a repeatedly presented odor and whether the mouse demonstrates dishabituation when presented a novel odor.

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the ...

Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as a useful biological indicator of water quality. Even though crayfish cannot directly specify the substances that cause water quality deterioration, they can immediately (within a few seconds) warn humans of water quality deterioration via acute changes in their cardiac and behavioral activities.
In this study, we present a noninvasive method that is simple enough to be implemented under various conditions due to a combination of simplicity and reliability in one model.
This approach, in which the biological organisms are implemented into environmental evaluation processes, provides a reliable and timely alarm for warning of and preventing acute water deterioration in an ambient environment. Therefore, this noninvasive system based on crayfish physiological and ethological parameter recordings was investigated for the detection of changes in an aquatic environment. This system is now applied at a local brewery for controlling&#160;quality of the water used for beverage production, but it can be used at any water treatment and supply facility for continuous, real-time water quality evaluation and for regular laboratory investigations of crayfish cardiac physiology and behavior.

This article presents a noninvasive biomonitoring system for the continuous recording and analyses of crayfish cardiac and locomotor activities. This system consists of a near-infrared optical sensor, a video-tracking module, and software for evaluating crayfish heartbeats that reflects its physiological condition and characterizes crayfish behavior during heartbeat fluctuations.

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as ...

Assessing the Autonomic and Behavioral Effects of Passive Motion in Rats using Elevator Vertical Motion and Ferris-Wheel Rotation

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and Ferris-wheel rotation devices. These assays can help confirm the integrity and normal functioning of the autonomic nervous system. They are coupled to quantitative measures based on defecation counting, open-field examination, and balance beam crossing. The advantages of these assays are their simplicity, reproducibility, and quantitative behavioral measures. The limitations of these assays are that the autonomic reactions could be epiphenomena of non-vestibular disorders and that a functioning vestibular system is required. Examination of disorders such as motion sickness will be greatly aided by the detailed procedures of these assays.

Protocols are presented to assess the autonomic and behavioral effects of passive motion in rodents using elevator vertical motion and Ferris-wheel rotation.

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and ...

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...

Tactile Semiautomatic Passive-Finger Angle Stimulator (TSPAS)

Passive tactile perception is the ability to passively and statically perceive stimulus information coming from the skin; for example, the ability to sense spatial information is the strongest in the skin on the hands. This ability is termed tactile spatial acuity, and is measured by the tactile threshold or discrimination threshold. At present, the two-point threshold is extensively used as a measure of tactile spatial acuity, although many studies have indicated that critical deficits exist in two-point discrimination. Therefore, a computer-controlled tactile stimulus system was developed, the tactile semiautomated passive-finger angle stimulator (TSPAS), using the tactile angle discrimination threshold as a new measure for tactile spatial acuity. The TSPAS is a simple, easily operated system that applies raised angle stimuli to a subject&#39;s passive fingerpad, while controlling movement speed, distance, and contact duration. The components of the TSPAS are described in detail as well as the procedure to calculate the tactile angle discrimination threshold.

Tactile Semiautomatic Passive-Finger Angle Stimulator TSPAS

Presented is the tactile semiautomated passive-finger angle stimulator TSPAS, a new way to assess tactile spatial acuity and tactile angle discrimination using a computer-controlled tactile stimulus system that applies raised angle stimuli to a subject&#39;s passive fingerpad, while controlling for movement speed, distance, and contact duration.

Passive tactile perception is the ability to passively and statically perceive stimulus information coming from the skin; for example, the ability to ...

Chronic Stress Shifts Effort-Related Choice Behavior in a Y-Maze Barrier Task in Mice

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that measures motivation to expend effort and obtain reward. In mice, chronic stress exposure significantly impacts motivation to work for a higher value reward when a lesser value reward is freely available compared to unstressed mice. Here we describe the chronic corticosterone administration paradigm, which produces a shift in effortful responding in the Y-maze barrier task. In the Y-maze task, one arm contains 4 food pellets, while the other arm contains only 2 pellets. After mice learn to select the high reward arm, barriers with progressively increasing height are then introduced into the high reward arm over multiple test sessions. Unfortunately, most chronic stress paradigms (including corticosterone and social defeat) were developed in male mice and are less effective in female mice. Therefore, we also discuss chronic non-discriminatory social defeat stress (CNSDS), a stress paradigm we developed that is effective in both male and female mice. Repeating results with multiple distinct chronic stressors in male and female mice combined with increased usage of translationally relevant behavior tasks will help to advance the understanding of how chronic stress can precipitate mood disorders.

The Y-maze barrier task is a behavior test that examines motivation to expend effort for reward. Here, we discuss testing multiple well-validated chronic stressors including chronic corticosterone and social defeat stress with this behavior, as well as the novel chronic non-discriminatory social defeat stress (CNSDS), which is effective in females.

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that ...