A. M. was partially funded by the Vicerrector&#237;a de Docencia, Benem&#233;rita Universidad Aut&#243;noma de Puebla. We are especially indebted to the staff of the animal facility &#8220;Claude Bernard&#8221; for the use of the rats for the shoot.&#160;We thank anonymous referees for their comments on early versions of this manuscript. The presentation is less strident and more balanced because of their thoughtful comments

The experimental protocols and animal husbandry were conducted in accordance with institutional guidelines.
1. Materials
<ol>
	<li>Find a complete list of the materials used to implement the method in the Table of Materials. Use Figure 1 and seek expert advice to construct an inverted T-shaped table, observation cages, and cage dividers. Follow the safety indications for the use of sharp tools and potentially dangerous materials.</li>
	<li>Make an inverted T-shaped table by gluing two rail-like wooden bars (45 cm length, separated 0.6 cm from each other) onto the top and in the middle of a piece of wood (100 cm x 45 cm x 1.5 cm). Then, place a second piece of wood (50 cm x 45 cm x 0.6 cm) vertically between the rail-like bars. Ensure that this second piece of wood prevents the pair of rats on one side from seeing the other pair on the opposite side (Figure 1).</li>
	<li>Use glass and acrylic to create eight observation cages. Ensure that each cage (19 cm wide, 19 cm long, 10 cm high, 3 mm thick) has 24 equidistant holes (5 mm diameter) arranged in three rows in the middle of one of its sides. Make this side of acrylic (3 mm thick) and shorten the height of the opposite side by 0.7 cm to allow the rat to breathe.
	<ol>
		<li>Make sure each observation cage has a sliding lid made of acrylic to prevent the rat from rearing and distracting itself during the 60 min observation period.</li>
	</ol>
	</li>
	<li>Make four dividers out of acrylic (19 cm wide, 30 cm high, 3 mm thick). Drill 24 holes (5 mm diameter) that match those in the observation cages, each in one clear divider and one opaque divider.</li>
	<li>Prepare data sheets in advance to record the occurrence of yawns. Include, in the heading of each data sheet, the name of the observer, experimental condition (i.e., familiar or unfamiliar rats), relevant test situation (OC, VC, VOC, NVOC), the date, and initial and final times of the observation. Divide the rest of the data sheet into two columns, each with the number of the rat written in the upper part, to record the yawning behavior of the rats.</li>
</ol>
2. Procedure
<ol>
	<li>House 144 male rats in groups of four in plastic cages (home cages) from weaning until they reach 2.5 months in age. Do not use female rats, because they tend to show greater variation in behavior due to hormonal cycles, which may be confused with the effects of the test situation. Make sure that the rats in each cage are not siblings. 
	NOTE: It might be difficult to obtain all the rats together. In that case, create blocks of at least 8 familiar rats and 8 unfamiliar rats so that every test situation is represented once in each experimental condition30.</li>
	<li>To identify the rats, mark them with symbolic numbers on the tails using a commercial marker. For example, represent the numbers 1 to 4 by combining dots and lines (e.g. ,one dot for number 1 and one line for number 4). Identify familiar and unfamiliar rats using different colors. 
	NOTE: Handle the rats following safety directions provided by animal facility staff, and comply with recommendations concerning the correct use of laboratory animals at the institution where experiments will be performed.</li>
	<li>Randomly choose which home cages will house the group of familiar rats and which will house the unfamiliar group. For example, suppose there are 16 available rats living in four home cages: number the home cages 1 to 4, then use R (download it at&#160;http://cran.r-project.org/) as follows [after the prompt (&#62;)]: 
	&#62; sample(4,4) 
	[1] 4 3 2 1
	<ol>
		<li>Group the familiar (or unfamiliar) rats in home cages 4 and 3, and group the unfamiliar (or familiar) rats in home cages 2 and 1. Make sure the animal facility staff maintains the identity of each cage and handles the rats in the same manner as all other rats in the animal facility.</li>
		<li>Use the R program again and randomly choose rats to form each pair in each experimental condition. Ensure that the rats in each pair of familiar rats originate from the same home cage, and ensure that the opposite is true for the pairs in the unfamiliar group. Randomly choose pairs of rats for each test situation in each experimental condition.</li>
	</ol>
	</li>
	<li>Perform two test sessions per day with two of the eight possible test situations (four for each familiar and unfamiliar rats) per test session (i.e., 60 min observation period). Conduct the two test sessions consecutively within 3 h. 
	NOTE: Make sure to conduct all test sessions (four pairs of rats per day) either in the morning or afternoon to avoid any confounding factors. Run a complete replicate (eight test situations) of the experiment over two consecutive days.
	<ol>
		<li>Make sure to use the test situations in a random sequence for each replicate of the experiment. For example, use numbers 1 to 8 to identify each test situation, then use R as follows: 
		&#62;sample(8,8) 
		[1] &#8203;8 7 4 6 5 1 2 3</li>
		<li>Allocate test situations 8 and 7 to test session 1, test situations 4 and 6 to test session 2, and so on. Then, randomly choose the side of the inverted T-shaped table where each pair of rats (test situation) will be placed.</li>
	</ol>
	</li>
	<li>Repeat the same procedure (steps 2.3 to 2.4.2) for a second replicate (i.e., block) of the experiment. 
	NOTE: If the aim of the study is to test the effects of a social condition (i.e., two interacting rats) on yawning frequency, use one rat placed in an observation cage next to an empty observation cage as a control for each of the four test situations. Perform this experiment 2x for each test situation for a control group of eight rats.</li>
	<li>Set up the test session by transferring the first four rats from the animal facility to the observation room, where they will remain for 15 min to acclimate to the novel environment. Transport the rats in individual cages and keep them separated from each other during transportation and in the observation room. 
	NOTE: Follow the safety directions for transporting animals provided by the animal facility staff and use the indicated clothes to work with animals in the laboratory. The rats will not have access to food and water while they are in the observation room.</li>
	<li>After the acclimatization period has passed, place the inverted T-shaped table on a larger rectangular table. Make sure there is a ceiling lamp that sufficiently lights the room when making observations.</li>
	<li>Put filter paper on the bottom of each observation cage and place the cages in pairs on each side of the inverted T-shaped table. Position the corresponding divider in between each pair of cages. 
	NOTE: The filter paper makes it easier to clean the cages and provides a rough surface on which the rats can move without slipping.</li>
	<li>Strategically place two digital camcorders such that each records the yawning behavior of each pair of rats. Make sure the camcorders are safely fixed to tripods and correctly orientated to the observation cages. Connect the camcorders to a desktop computer to simultaneously monitor the behavior of the rats. 
	NOTE: Store the digital information on flash drives to permanently archive the experimental sessions. Use flash drives with a high storage capacity.</li>
	<li>Following the acclimatization period, place the rats in the observation cages according to the allocation previously determined. Set the automatic focus of the camcorder and start the video recording simultaneously with a stopwatch. Stop the video recording at the end of the 60 min observation period. 
	NOTE: While the experimenters may be outside of the room during the test session and observing remotely, it is recommended that one person is in the observation room (as far away as possible from the experimental setting) while monitoring the behavior of the rats to ensure that the test session proceeds without interruption.</li>
	<li>When the observation is over, return the rats to their home cages in the animal facility. Ask the animal facility staff to ensure that the rats have access to food and water again. Thoroughly clean the observation cages using a nontoxic detergent and prepare the experimental set-up to perform the second and final test sessions of the day. 
	NOTE: Clean the wood to remove any scents left by the rats when placed in the observation cages, which could affect the behavior of the next pair of rats tested.</li>
	<li>Train one to two volunteers partially blind to the assigned treatments to identify and record yawning using an all-occurrence sampling method. Make sure the observers use the definition of yawning described in the introduction section.</li>
	<li>Play back and project each video on a computer screen using any standard playback system. Ask the observer to view each video and observe and record yawning behavior using the previously prepared data sheets, then allow him/her to review the videos at slower speeds to enhance the ability to observe and measure yawning.
	<ol>
		<li>Ask the observers to score yawning using a notation system similar to the following: use vertical lines with the minute written as a superscript to represent the occurrence of a yawn (e.g., |3 ||6 |9, indicating one yawn at minute 3, two yawns at minute 6, and one yawn at minute 9). Score yawning as a sequence (e.g.,1.2, 2.2, 3.2, 5.0, 5.8) to record it with greater precision (minutes rounded to one decimal place).</li>
		<li>Alternatively, directly input data from the videos into a computer using standard data collection programs. Make sure the observer is acquainted with such programs. 
		NOTE: If necessary, allow the observer to view each video in more than one session. Ensure that one observer views all the videos corresponding to one block of experiments. If two different observers viewed these videos, there may be a risk that differences in their abilities to observe and record yawning will confound the effects of the test situations. It is important to score the intra-observer reliability between different observers on 10%-20% of the videos to ensure that yawning is annotated in the same way.</li>
	</ol>
	</li>
</ol>
3. Data processing
<ol>
	<li>Transcribe the temporal sequence of yawns in each rat from the data sheets to a spreadsheet. Ensure there is one column for each rat with a short heading (e.g., fr1.l and fr1.r to indicate the first pair of familiar rats on the left and right sides, respectively). Ensure that all columns are the same length by filling the empty cells with either 0&#160;or NA (see below). 
	NOTE: Use the general guide provided as a <a href="https://www.jove.com/files/ftp_upload/59289/Supplemental_Material.zip" target="_blank">supplementary document</a> to fully understand the following steps to process the data, measure contagious yawning, and obtain contagious yawning curves.
	<ol>
		<li>If yawning was recorded to the nearest minute, type in the number of yawns at each relevant minute and use 0 (no yawn) to fill in the cells when no yawn occurred in a given minute. Save the worksheet as a text file (.txt).</li>
		<li>If yawning was recorded to the nearest decimal of a minute, type in the sequence top down and use &#34;NA&#34; to fill in the empty spaces of columns (rats) in which the numbers of yawns (rows) is lower than that of the column with the maximum number (rows) of yawns recorded for a rat. Save the worksheet as a text file (.txt). 
		NOTE: As R has the ability to work with empty cells, the worksheet can be saved with columns of different lengths. Use the help options provided by R to handle missing data.</li>
	</ol>
	</li>
	<li>Initiate R. Import the data from the file in which they were previously located.</li>
	<li>Save the program codes with the extension &#34;.R&#34; (provided as <a href="https://www.jove.com/files/ftp_upload/59289/Supplemental_Material.zip" target="_blank">supplementary material</a>), then download the specific program code (see general guide) depending on whether yawns were recorded as integers or fractional numbers. Run the program for each pair of rats and desired number of time frames.
	<ol>
		<li>Run the program again, now using a random distribution of the number of yawns from each rat (see general guide). Follow the same steps (steps 3.3 to 3.3.1) for all experimental conditions (i.e., familiar and unfamiliar rats) and/or all the test situations. Proceed as indicated in the general guide and export the results to Excel. 
		NOTE: Instead of importing a previously saved program, as suggested above using a text editor, copy and directly paste the program into R&#39;s workspace.</li>
	</ol>
	</li>
	<li>To create yawn contagion curves, use the data previously saved in an spreadsheet. Subtract the non-contagion rates from the contagion rates for each pair of rats, time window, and test situation. Separate the analyses of the observed data and artificial (randomly distributed) data.
	<ol>
		<li>Next, calculate confidence intervals (CI) for each time window and test situation using a bootstrap procedure (see the general guide). Separate the analyses for familiar and unfamiliar rats. Then, combine the artificial data from the four test situations for each experimental condition and calculate the CI for each time window using a bootstrap procedure.</li>
		<li>Next, create plots for each test situation and experimental condition (see Figure 2 and Figure 3). Finally, perform a multiple regression analysis to compare the intensity of yawn contagion between test situations (see below).</li>
	</ol>
	</li>
</ol>

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The authors declare no conflicts of interest.

There are critical steps in the method that should be taken into account to obtain successful results. Familiar rats must share home cages for at least 1.5 months after weaning and before running the experiments. However, unfamiliar rats must live in separate home cages. In both cases, the pairs of rats must come from different litters but be as similar in age as possible. Regarding the observation cages, their holes should match those in the dividers, because this is the only way to guarantee olfactory contact between t...

Traditionally, communicatory behavior has been studied from two perspectives. From one perspective, ethologists observe and record the behavior of animals in natural settings and attempt to recognize its adaptive value1. The particular sense or senses involved have not been the primary interest of these studies. From another perspective, physiologists are more interested in unraveling the mechanisms by which animals communicate1; hence, laboratory studies have provided methods to address the role that sensory modalities play in communication2,3. These two perspectives are indeed complementary, because knowledge of both adaptive value and immediate mechanisms is necessary to gain a comprehensive understanding of communicatory behaviors in the social life of animals.
Yawning behavior is a conspicuous component of the behavioral repertoire in several species of vertebrates4, ranging from fish to primates5. It can be described as a slow opening of the mouth and maintenance of its open position, followed by a more rapid closure of the mouth5. The duration of the whole sequence depends on the species; for example, primates yawn for longer durations than non-primate species6. In many species, with humans being the exception, males tend to yawn more frequently than females7. This feature might underpin the possible communicatory function of yawning, although regular patterns of yawning and its daily frequency may also suggest a physiological function. In rats, spontaneous yawning follows a circadian rhythm, with peaks of high frequency occurring in the morning and afternoon8,9.
One interesting feature of yawning behavior is that it can be a contagious act (when the releasing stimulus of a behavior happens to be another animal behaving in the same way10) in several species of vertebrates11,12,13,14,15,16, including birds17 and rodents18. Furthermore, recent evidence has indicated that contagious yawning may reflect a communicatory role, because the yawning of one rat can affect the physiological state of another when exposed to olfactory cues19. However, whether or not yawning has a communicatory role is still under debate20,21, and analyzing contagious yawning is an essential first step to solve this issue.
On the other hand, contagious yawning has been linked to an animal&#39;s ability to empathize with the perspectives of other animals; hence, closely related individuals are more likely to show contagion4. This hypothesis has been frequently tested in laboratory conditions in which animals are presented with yawn stimuli on video12,13; hence, contagion can only occur through visual cues. Other investigations have assessed yawn contagion in more natural conditions using groups of animals14,15. A major problem of this is that socially interacting animals often respond to cues and exchange signals that are conveyed through combinations of sensory modalities. Disentangling the actual senses involved in a given behavior from their combined effects is not always an easy task. Typically, researchers pharmacologically or surgically hinder an animal&#39;s use of a given sense, then infer the role of that sense in the relevant behavior2,3,18,22. Fortunately, there are other methods in which only physical barriers are used to either allow or impede communication between animals23,24,25, thereby achieving greater biological validity.
The method proposed here has been specifically designed to study contagious yawning in familiar and unfamiliar rats in a social setting. According to the empathetic hypothesis, the former group should be more susceptible to contagious yawning. The method does not require the animals to be surgically or pharmacologically deprived of any senses. Instead, it works by placing the rats in adjacent cages with holes and physically obstructing their communication using either clear or opaque dividers with or without holes. Thus, four test conditions can be examined: (1) olfactory communication (OC, perforated opaque divider), (2) visual communication (VC, nonperforated clear divider), (3) visual and olfactory communication (VOC, perforated clear divider), and (4) neither visual nor olfactory communication (NVOC, nonperforated opaque divider). Therefore, researchers can compare the relative contributions of olfactory, visual, and to some extent, auditory cues in yawn contagion. This approach is not new, as similar methods have been used to isolate the senses involved in certain communicatory behaviors in animals such as lizards23 and mice26. In fact, Gallup and colleagues27 have used a similar method to demonstrate the role of visual cues in contagious yawning in budgerigars. The main features of these methods are simulation of a social context and the minimal stress inflicted on the animals. Furthermore, the use of interacting animals increases the biological validity of the conclusions.
There are several ways to measure contagious yawning25,28. Dr. Stephen E. G. Lea (personal communication, 2015) helped us numerically adapt a method previously employed by primatologists13,14 for an earlier analysis of the data used here18. Presented in this protocol is an enhanced version of this method with a wider range of applications. It consists of weighting the total number of a rat&#39;s yawns, within and outside of a given time window, by the proportion of observation time corresponding to the yawns within and outside the time window.
For example, if it is assumed that rats A and B are observed for 12 min, their yawning is recorded to the nearest minute, and a 3 min time window is set to measure contagious yawning. Next, the following sequences of yawns for each of those rats are considered: rat A (0,0,0,1,0,0,2,0,0,0,2,1) and rat B (0,1,1,0,1,1,0,0,0,0,0,3). It should be noted that each number (0-3) corresponds to the number of yawns scored at each min. For rat A, during minutes 1, 10, and 11 (numbers in bold type), rat B does not yawn within the preceding 3 min (the chosen time window) or within that minute. In those minutes, rat A yawns a total of 2 times. Therefore, the yawn rate of rat A without any yawn stimulus (non-post-yawn yawn rate) is 2/3 (i.e., 0.67 yawns/min). In the remaining 9 min, rat B yawns at least one time in either the same minute or the 3 previous minutes. Rat A yawns a total of four times in those 9 min. Therefore, the yawn rate of rat A in response to a yawn stimulus (post-yawn yawn rate) is 4/9 (i.e., 0.44 yawns/min). The application of the same procedure to rat B yields a non-post-yawn yawn rate of 2/3 (i.e., 0.66) and post-yawn yawn rate of 5/9 (0.55).
On the other hand, if yawning is recorded to the nearest decimal of a minute, yawn contagion will result in an adjusted post-yawn time. For example, if the following yawn times are recorded over a 12 min observation period for rats A and B: rat A (2.3, 5.1, 5.8, 10.4, 10.8, 11.1) and rat B (1.2, 2.4, 4.5, 5.1, 11.2, 11.6, 11.8). For rat A, the time periods over which rat B does not yawn within the past 3 min range from 0 to 1.2 min and from 8.1 to 11.2 min (i.e., 3.1 min), which yields a total of 4.3 min of non-post-yawn time. The number of times that rat A yawns during those times is three (numbers in bold type), so the non-post-yawn yawn rate is 3/4.3 (i.e., 0.69), while the post-yawn yawn rate is 3/7.7 (i.e., 0.38; the denominator from 12-4.3 min). Similarly, for rat B, the time periods over which rat A does not yawn within the past 3 min range from 0 to 2.3 min and from 8.8 to 10.4 min, which yields a total of 3.9 min. The number of times rat B yawns within those periods is one, so the non-post-yawn yawn rate is 1/3.9 (i.e., 0.25). Accordingly, the post-yawn yawn rate is 6/8.1 (i.e., 0.74).
While a near-contemporaneous match in behavior is an ideal criterion to demonstrate the presence of a contagion, aspects such as the constraints on what an individual attends to, time of reaction to a stimulus, distribution of the behavior over time (e.g., yawning may occur in episodes), and time to acclimatize to the experimental setting all give rise to species differences, making it difficult to use a unique time window. This may be the reason why researchers have used time windows that vary from seconds5 to several minutes11, which creates problems when comparing results28. Because of this, it is proposed to repeat the procedure described above for a range of time windows to obtain yawn contagion curves and compare the yawn contagion curves between species.
Equivalent yawn contagion curves can be compared by randomly distributing the number of yawns observed for each rat over the observation period. Thus, the proposed method to measure yawn contagion offers two types of controls: the (1) yawn rate occurring outside of the time window (non-post-yawn time) and (2) artificial yawn contagion curve obtained from the random distribution of the number of yawns. Therefore, this approach to analyze yawn contagion is a step forward from other procedures, such as those comparing the percentage or frequency of yawning within a single time window to that occurring outside this window25, without taking into account the actual times frames. The method is complemented by an R-based program29 to conveniently and objectively calculate the probability of contagious yawning for one or more time windows.
To illustrate the usefulness of this method and advantages of the R-based program, a data set from a previously published study18 is used. The experimental condition consisted of 144 male rats allocated to either a familiar or unfamiliar condition. The rats in each experimental condition were subdivided into four subgroups of nine pairs and exposed to any of the four test situations described above. The yawning behaviors of the rats in each experimental condition and test situation were then recorded over a period of 60 min.

<table><tbody><tr><td>Acrylic dividers</td><td>Handcrafted</td><td>Not available</td><td>Two dividers, one clear and one opaque, will have 24 holes each. The other two dividers, one clear and one opaque, will have no holes. See the main text for details of construction.</td></tr><tr><td>An R-based program</td><td>Benem&amp;eacute;rita Universidad Aut&amp;oacute;noma de Puebla</td><td>Not available</td><td>This is the program used to assess yawn contagion in rats. See the main text for information about the way the program is used.</td></tr><tr><td>Data sheets</td><td>The user can elaborate them</td><td>Not available</td><td>These forms will be used for the observer to record the frequency of yawning behaviour by viewing the video recordings. Alternatively, a notebook can be use provided you follow the suggestions given in the main text.</td></tr><tr><td>Desktop computer</td><td>Any maker</td><td>Not available</td><td>Make sure the computer has a video card capable of conveniently processing the video recordings of yawning behaviour.&amp;nbsp;</td></tr><tr><td>Digital camcorders</td><td>Any maker</td><td>Not available</td><td>They will be used to video record yawning behaviour of each pair of rats; there will be 2 pairs of rats per experimental session.&amp;nbsp;</td></tr><tr><td>Flash drive</td><td>Any maker</td><td>Not available</td><td>Each experimental session will last 60 min, and so you will require sufficient memory to store the video recording.</td></tr><tr><td>Glass cages</td><td>Handcrafted</td><td>Not available</td><td>Each cage (19 X 19 X 10 cm height) will have 24 holes (0.5 cm diameter) forming three rows in the middle of one of its sides. See the main text for more details about their construction. It is recommended to fabricate one extra cage in case one of them is accidentally broken.&amp;nbsp;</td></tr><tr><td>Markers</td><td>Sharpie or any other maker</td><td>Not available</td><td>Permanent markers to number the rats. See the main text to see one way of using painting symbols on the rat&#039;s tail.&amp;nbsp;</td></tr><tr><td>Pencils</td><td>Any maker</td><td>Not available</td><td>They are used by the observer to record the frequency of yawning. It is important that the observer has previously been trained to recognize yawning behaviour and operate the video player system.&amp;nbsp;</td></tr><tr><td>R software</td><td>R Development Core Team</td><td>Not available</td><td>Download R at: http://cran.r-project.org/&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Rail-like wooden bars</td><td>Handcrafted</td><td>Not available</td><td>They will be fixed in the middle of the rectangular wooden sheet&amp;nbsp; forming a track, where a second wooden sheet is placed. See the main text for additional instructions for construction.</td></tr><tr><td>Rectangular table</td><td>Any maker</td><td>Not available</td><td>This is the table (approximately 2 x 1 m) where the inverted T-shaped table will be placed for performing the observation of yawning behaviour.</td></tr><tr><td>Sprague-Dawley male rats</td><td>Any local supplier of laboratory animals</td><td>Not available</td><td>Nine pairs of male rats per test situation are necessary for each group, familiar and unfamiliar rats, because with this sample size the interindividual variation that might exist in yawning frequency will not severely affect the conclusions drawn from the statistical analysis performed to the data.&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Spreadsheet software</td><td>Microsoft</td><td>Not available</td><td>Excel will be the software used to store the yawning recordings initially recorded on the data sheets. Revise the main text for instructions about the recommended way of doing the transcription.</td></tr><tr><td>Square filter papers</td><td>Any maker</td><td>Not available</td><td>They are used for covering the cage&#039;s bottom.</td></tr><tr><td>Tripods</td><td>Any maker</td><td>Not available</td><td>They will be used for fixing the camcorders in front of each pair of observation cages.</td></tr><tr><td>Wooden Inverted T-shaped table</td><td>Handcrafted</td><td>Not available</td><td>Read the instructions in the main text to see the way of constructing it. If preferred, a different material to wood can be used. Make sure any material is as resistant as possible to the transmission of ultrasounds, which the rats might use for communication.&amp;nbsp;&amp;nbsp;</td></tr></tbody></table>

a laboratory method to measure contagious yawning in rats

Communication is an essential aspect of animal social life. Animals may influence one another and come together in schools, flocks, and herds. Communication is also the way sexes interact during courtship and how rivals settle disputes without fighting. However, there are some behavioral patterns for which it is difficult to test the existence of a communicatory function, because several types of sensory modalities are likely involved. For example, contagious yawning is a communicatory act in mammals that potentially occurs through sight, hearing, smell, or a combination of these senses depending on whether the animals are familiar to one another. Therefore, to test hypotheses about the possible communicatory role of such behaviors, a suitable method is necessary to identify the participating sensory modalities.
The method proposed here aims to obtain yawn contagion curves for familiar and unfamiliar rats and evaluate the relative participation of visual and olfactory sensory modalities. The method uses inexpensive materials, and with some minor changes, it can also be used with other rodent species such as mice. Overall, the method involves the substitution of clear dividers (with or without holes) with opaque dividers (with or without holes) that either allow or prevent communication between rats placed in adjacent cages with holes in adjoining sides. Accordingly, four conditions can be tested: olfactory communication, visual communication, both visual and olfactory communication, and neither visual nor olfactory communication. As social interaction occurs between the rats, these test conditions simulate what may occur in a natural environment. In this respect, the method proposed here is more effective than traditional methods that rely on video presentations whose biological validity can raise concerns. Nonetheless, it does not discriminate between the potential role of hearing and roles of smell and vision in yawn contagion.

The rats were selected from a previously produced sub-line of Sprague-Dawley rats that were selected for frequent yawning (approximately 22 yawns per hour31). However, the nine pairs of unfamiliar and nine pairs of familiar male rats (between 2.5 and 3 months of age) used per test situation yawned approximately 12 times per hour, on average18. Therefore, the test situations to measure yawn contagion partially inhibited yawning behavior.
...

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A Laboratory Method to Measure Contagious Yawning in Rats

The method described here aims to obtain yawn contagion curves in pairs of familiar or unfamiliar male rats. Cages with holes separated by either clear or opaque partitions with (or without) holes are used to detect whether visual, olfactory, or both types of sensory cues can stimulate yawn contagion.

Communication is an essential aspect of animal social life. Animals may influence one another and come together in schools, flocks, and herds. ...

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7.0K Views.  Benemérita Universídad Autónoma de Puebla Pue. Mexico. Ons protocol is belangrijk omdat het een gemakkelijke manier biedt om te onderzoeken welke zintuigen betrokken kunnen zijn bij besmettelijk geeuwen bij ratten. Het gebruik van een niet-invasieve procedure en de beschikbaarheid van de applicatiesoftware, die de berekening voor het meten van besmettelijke geeuwen versnelt, zijn de belangrijkste voordelen van deze techniek. Personen die nog nooit hebben uitgevoerd deze techniek mag niet worden geconfronteerd met grote problemen in de uitvoering,...

Video: A Laboratory Method to Measure Contagious Yawning in Rats

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Neuroscience

A New Method for Inducing a Depression-Like Behavior in Rats

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is no existing format for studying the mechanism of action, prevention, containment, and treatment of contagious depression. The purpose of this study, therefore, was to establish the first animal model of contagious depression.
Healthy rats can contract depressive behaviors if exposed to depressed rats. Depression is induced in rats by subjecting them to several manipulations of chronic unpredictable stress (CUS) over 5 weeks, as described in the protocol. A successful sucrose preference test confirmed the development of depression in the rats. The CUS-exposed rats were then caged with na&#239;ve rats from the contagion group (1 na&#239;ve rat/2 depressed rats in a cage) for an additional 5 weeks. 30 social groups were created from the combination of CUS-exposed rats and na&#239;ve rats.
This proposed depression-contagion protocol in animals consists mainly of cohabiting CUS-exposed and healthy rats for 5 weeks. To ensure that this method works, a series of tests are carried out - first, the sucrose preference test upon inducing depression to rats, then, the sucrose preference test, alongside the open field and forced-swim tests at the end of the cohabitation period. Throughout the experiment, rats are given tags and are always returned to their cages after each test.
A few limitations to this method are the weak differences recorded between the experimental and control groups in the sucrose preference test and the irreversible traumatic outcome of the forced swim test. These may be worth considering for suitability before any future application of the protocol. Nonetheless, following the experiment, na&#239;ve rats developed contagion depression after 5 weeks of sharing the same cage with the CUS-exposed rats.

This protocol describes a new model by which healthy rats could contract depression over a given time periodthrough contagion by exposure to chronic unpredictable stressed (CUS) rats.

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is ...

Tickling, a Technique for Inducing Positive Affect When Handling Rats

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk for both the animals and the hands of their handlers, decreased animal welfare, and less valid research data. To minimize negative effects on experimental results and human-animal relationships, research animals are often habituated to being handled. However, the methods of habituation are highly variable and often of limited effectiveness. More potently, it is possible for humans to mimic aspects of the animals&#39; playful rough-and-tumble behavior during handling. When applied to laboratory rats in a systematic manner, this playful handling, referred to as tickling, consistently gives rise to positive behavioral responses. This article provides a detailed description of a standardized rat tickling technique. This method can contribute to future investigations into positive affective states in animals, make it easier to handle rats for common husbandry activities such as cage changing or medical/research procedures such as injection, and be implemented as a source of social enrichment. It is concluded that this method can be used to efficiently and practicably reduce rats&#39; fearfulness of humans and improve their welfare, as well as reliably model positive affective states.

This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk ...

Noninvasive, High-throughput Determination of Sleep Duration in Rodents

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a long recovery period. To perform an EEG recording, the animal is connected to a receiver, creating an unnatural tether to the head-mount. EEG monitoring is time consuming, carries risk to the animal, and is not a completely natural setting for the measurement of sleep. Alternative methods to detect sleep, particularly in a high-throughput fashion, would greatly advance the field of sleep research. Here, we describe a validated method for detecting sleep via activity-based home-cage monitoring. Previous studies have shown that sleep assessed via this method has a high degree of agreement with sleep defined by traditional EEG-based measures. Whereas this method is validated for total sleep time, it is important to note that sleep bout duration should be assessed by an EEG which has better temporal resolution. The EEG can also differentiate rapid eye movement (REM) and non-REM sleep, giving more detail about the exact nature of sleep. Nevertheless, activity-based sleep determination can be used to analyze multiple days of undisturbed sleep and to assess sleep as a response to an acute event (like stress). Here, we show the power of this system to detect the response of mice to daily intraperitoneal injections.

We describe a high-throughput method of measuring sleep by means of activity-based home-cage monitoring. This method offers advantages over traditional EEG-based methods. It is well validated for the determination of total sleep duration and can be a powerful tool to monitor sleep in rodent models of human disease.

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a ...

Using Clicker Training and Social Observation to Teach Rats to Voluntarily Change Cages

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on well-being and can affect the comparability and reproducibility of research results, the amount of stress experienced by laboratory animals should be minimized and avoided when possible. Further, the direct contact between the rat and animal caretaker during the cage change bears hygiene risks and therefore possibly negatively impacts the well-being of the rats and the quality of the research. 
Our protocol aims to improve the routinely performed cage changing procedure. For this reason, we present a feasible protocol that enables rats to learn via clicker training and observation to voluntarily change to a clean cage. This training helps to reduce stress caused by the physical disturbance and handling associated with the cage changes and concurrently enables a reduction in direct contact between animal and animal caretaker after the training phase is completed.
The implementation of clicker training to rats is fast and easy. Rats are generally interested in the training and efficiently learn the desired behavior, which entails changing cages through a pipe. Even without training, the rats learn to perform the desired behavior by observation, as 80% of the observational learning group successfully changed cages when tested. The training further helps to establish a relationship of trust between trainer and animal. As hygiene and well-being are both very important in animal experiments, this protocol might also help to improve high-quality research.

This protocol introduces a method of cage change for rats via clicker training. Rats learn the desired behavior not only by direct training but also by observational learning. The implementation of this fast and easy protocol might help to improve well-being and hygiene in rodent facilities.

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Immunology and Infection

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease and arrhythmias in people with epilepsy (i.e., epileptic heart.) Additionally, cardiac and autonomic disturbances have been reported surrounding seizures. 1:1,000 epilepsy patients/year die of sudden unexpected death in epilepsy (SUDEP). The mechanisms for SUDEP remain incompletely understood. Electroencephalograms (EEG) and electrocardiograms (ECG) are two techniques routinely used in the clinical setting to detect and study the substrates/triggers for seizures and arrhythmias. While many studies and descriptions of this methodology are in rodents, their cardiac electrical activity differs significantly from humans. This article provides a description of a non-invasive method for recording simultaneous video-EEG-ECG-oximetry-capnography in conscious rabbits. As cardiac electrical function is similar in rabbits and humans, rabbits provide an excellent model of translational diagnostic and therapeutic studies. In addition to outlining the methodology for data acquisition, we discuss the analytical approaches for examining neuro-cardiac electrical function and pathology in rabbits. This includes arrhythmia detection, spectral analysis of EEG and a seizure scale developed for restrained rabbits.

Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death.

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease ...

Medicine

Noninvasive Real-Time Whole-Body Plethysmography for Cough Sensitivity in Mice

Methods for Detecting Cough and Airway Inflammation in Mice

Chronic cough, which lasts for more than 8 weeks, is one of the most common complaints requiring medical attention, and patients suffer from a huge socioeconomic burden and a marked decrement in quality of life. Animal models can mimic the complex pathophysiology of the cough and are important tools for cough research. The detection of cough sensitivity and airway inflammation is of great significance for studying the complex pathological mechanism of cough. This article describes the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples (including blood, lung, spleen, and trachea) of mice. It introduces some methods to assess airway inflammation, including pathological changes in hematoxylin and eosin (HE)-stained lung and trachea sections, the total protein concentration, the uric acid concentration, and the lactate dehydrogenase (LDH) activity in the supernatant of bronchoalveolar lavage fluid (BALF), and the leukocytes and differential cell counts of BALF. These methods are reproducible and serve as valuable tools to study the complex pathophysiology of cough.

Here, we describe the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples of mice and introduce some methods to assess airway inflammation.

Chronic cough, which lasts for more than 8 weeks, is one of the most common complaints requiring medical attention, and patients suffer from a huge ...

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