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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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 border="0" cellpadding="0" cellspacing="0" style="width:860px;" width="860">
	<tbody>
		<tr height="83">
			<td height="83" style="height:83px;width:221px;">Acrylic dividers</td>
			<td style="width:173px;">Handcrafted</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">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 height="62">
			<td height="62" style="height:62px;">An R-based program</td>
			<td style="width:173px;">Benem&#233;rita Universidad Aut&#243;noma de Puebla</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">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 height="104">
			<td height="104" style="height:104px;">Data sheets</td>
			<td style="width:173px;">The user can elaborate them</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">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 height="62">
			<td height="62" style="height:62px;width:221px;">Desktop computer</td>
			<td style="width:173px;">Any maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">Make sure the computer has a video card capable of conveniently processing the video recordings of yawning behaviour.&#160;</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:221px;">Digital camcorders</td>
			<td style="width:173px;">Any maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">They will be used to video record yawning behaviour of each pair of rats; there will be 2 pairs of rats per experimental session.&#160;</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:221px;">Flash drive</td>
			<td style="width:173px;">Any maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">Each experimental session will last 60 min, and so you will require sufficient memory to store the video recording.</td>
		</tr>
		<tr height="125">
			<td height="125" style="height:125px;width:221px;">Glass cages</td>
			<td style="width:173px;">Handcrafted</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">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.&#160;</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:221px;">Markers</td>
			<td style="width:173px;">Sharpie or any other maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">Permanent markers to number the rats. See the main text to see one way of using painting symbols on the rat&#39;s tail.&#160;</td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;">Pencils</td>
			<td style="width:173px;">Any maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">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.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">R software</td>
			<td style="width:173px;">R Development Core Team</td>
			<td style="width:123px;">Not available</td>
			<td>Download R at: http://cran.r-project.org/&#160;&#160;</td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;width:221px;">Rail-like wooden bars</td>
			<td style="width:173px;">Handcrafted</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">They will be fixed in the middle of the rectangular wooden sheet&#160; forming a track, where a second wooden sheet is placed. See the main text for additional instructions for construction.</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:221px;">Rectangular table</td>
			<td style="width:173px;">Any maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">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 height="146">
			<td height="146" style="height:146px;width:221px;">Sprague-Dawley male rats</td>
			<td style="width:173px;">Any local supplier of laboratory animals</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">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.&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;width:221px;">Spreadsheet software</td>
			<td style="width:173px;">Microsoft</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">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 height="21">
			<td height="21" style="height:21px;width:221px;">Square filter papers</td>
			<td style="width:173px;">Any maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">They are used for covering the cage&#39;s bottom.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">Tripods</td>
			<td style="width:173px;">Any maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">They will be used for fixing the camcorders in front of each pair of observation cages.</td>
		</tr>
		<tr height="125">
			<td height="125" style="height:125px;">Wooden Inverted T-shaped table</td>
			<td style="width:173px;">Handcrafted</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">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.&#160;&#160;</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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6.9K Views.  Benemérita Universídad Autónoma de Puebla Pue. Mexico. 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.

Video: A Laboratory Method to Measure Contagious Yawning in Rats

The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden hand is touched. If the viewed and felt touches are given at the same time then this is sufficient to induce the compelling experience that the rubber hand is one's own hand. The RHI can be used to investigate exactly how the brain constructs distinct body representations for one's own body. Such representations are crucial for successful interactions with the external world. To obtain a subjective measure of the RHI, researchers typically ask participants to rate statements such as "I felt as if the rubber hand were my hand". Here we demonstrate how the crossmodal congruency task can be used to obtain an objective behavioral measure within this paradigm.
The variant of the crossmodal congruency task we employ involves the presentation of tactile targets and visual distractors. Targets and distractors are spatially congruent (i.e. same finger) on some trials and incongruent (i.e. different finger) on others. The difference in performance between incongruent and congruent trials - the crossmodal congruency effect (CCE) - indexes multisensory interactions. Importantly, the CCE is modulated both by viewing a hand as well as the synchrony of viewed and felt touch which are both crucial factors for the RHI.
The use of the crossmodal congruency task within the RHI paradigm has several advantages. It is a simple behavioral measure which can be repeated many times and which can be obtained during the illusion while participants view the artificial hand. Furthermore, this measure is not susceptible to observer and experimenter biases. The combination of the RHI paradigm with the crossmodal congruency task allows in particular for the investigation of multisensory processes which are critical for modulations of body representations as in the RHI.

We demonstrate how an objective measure can be employed in the widely employed rubber hand illusion paradigm. This measure is obtained by modifying the well-established crossmodal congruency task. This task allows the investigation of multisensory processes which are critical for modulations of body representations as in the rubber hand illusion.

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste reactivity with the behavioral aspects of operant self-administration of drugs. Under very brief general anaesthesia, rats are implanted with an intraoral (IO) cannula that allows delivery of test solutions directly in the oral cavity. Animals are then tested in operant self-administration chambers whereby they can press a lever to receive IO infusions of test solutions. IO self-administration has several advantages over experimental procedures that involve drinking a solution from a spout&#160;or operant responding for solid pellets or solutions delivered in a receptacle. Here, we show that IO self-administration can be employed to study self-administration of high fructose corn syrup (HFCS). Rats were first tested for self-administration on a progressive ratio (PR) schedule, which assesses the maximum amount of operant behavior that will be emitted for different concentrations of HFCS (i.e.&#160;8%, 25%, and 50%). Following this test, rats self-administered these concentrations on a continuous schedule of reinforcement (i.e.&#160;one infusion for each lever press) for 10 consecutive days (1 session/day; each lasting 3 hr), and then they were retested on the PR schedule. On the continuous reinforcement schedule, rats took fewer infusions of higher concentrations, although the lowest concentration of HFCS (8%) maintained more variable self-administration. Furthermore, the PR tests revealed that 8% had lower reinforcing value than 25% and 50%. These results indicate that IO self-administration can be employed to study acquisition and maintenance of responding for sweet solutions. The sensitivity of the operant response to differences in concentration and schedule of reinforcement makes IO self-administration an ideal procedure to investigate the neurobiology of voluntary intake of sweets.

The current study evaluates a novel procedure for assessing the reinforcing effects of palatable solutions in laboratory rats: intraoral self-administration. To this end, operant responding (i.e.&#160;lever pressing) for intraoral infusions of sweet solutions at different concentrations was measured on continuous and progressive ratios schedules of reinforcement.

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste ...

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

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 ...

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods ...

A Protocol of Manual Tests to Measure Sensation and Pain in Humans

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study demonstrates a quantitative sensory testing protocol using techniques to measure tactile sensation and pain threshold for pressure and heat using portable and easily accessed equipment. These techniques and equipment are ideal for new laboratories and clinics where cost is a concern or a limiting factor. We demonstrate measurement techniques for the following: cutaneous mechanical sensitivity on the arms and legs (von-Frey filaments), radiant and contact heat sensitivity (with both threshold and qualitative assessments using the Visual Analog Scale (VAS)), and mechanical pressure sensitivity (algometer, with both threshold and the VAS). The techniques and equipment described and demonstrated here can be easily purchased, stored, and transported by most clinics and research laboratories around the world. A limitation of this approach is a lack of automation or computer control. Thus, these processes can be more labor intensive in terms of personnel training and data recording than the more sophisticated equipment. We provide a set of reliability data for the demonstrated techniques. From our description, a new laboratory should be able to set up and run these tests and to develop their own internal reliability data.

The goal of this procedure is to demonstrate a battery of quantitative techniques for sensory and pain measurement in humans. The equipment and techniques described are commonly found in pain clinics or are easy to obtain.

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study ...

Psychophysical Tracking Method to Measure Taste Preferences in Children and Adults

The Monell two-series, forced-choice, paired-comparison tracking method provides a reliable measure of sweet taste preferences from childhood to adulthood. The method, which is identical for children, adolescents, and adults, is of short duration (&#60; 15 min), does not rely on sustained attention or place demands on memory (which would yield spurious age differences), and minimizes the impact of language development, making this method amenable to the cognitive limitations of pediatric populations. In this whole-mouth tasting method, subjects are asked to taste (without swallowing) pairs of solutions of different sucrose concentrations and to point to the solution they prefer. Each subsequent pair contains the participant&#39;s preceding preferred concentration and an adjacent stimulus concentration. The procedure continues until the subject chooses either a given concentration of sucrose when paired with both a higher and a lower concentration, or the highest or lowest concentration two consecutive times. Subjects are prevented from reaching response criteria on the basis of first or second position bias by the two-series design of the method, which counterbalances the order of solution presentation within each pair between the series (the weaker concentration is presented first in Series 1, second in Series 2). The geometric mean of the two sucrose concentrations chosen in Series 1 and 2 is an estimate of the participant&#39;s most preferred level of sucrose. Sucrose preference as determined with this laboratory-based measure has been shown to be associated with preference for sugars in foods and beverages and with taste receptor genotype, family history of alcoholism, and race/ethnicity, as well as depressive symptomatology among pediatric populations. The method has real-world relevance and has been applied to determine most preferred level of other tastes (e.g., salt), making it a valuable psychophysical tool.

Sweet taste has powerful hedonic appeal among people of all ages, particularly children. Described herein is a reliable and valid method that can be used to determine the level of sweetness most preferred, making it a valuable psychophysical tool for scientists.

The Monell two-series, forced-choice, paired-comparison tracking method provides a reliable measure of sweet taste preferences from childhood to ...

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, few studies have investigated the long-term effects of injury in rodent models of TBI. Shown here are behavioral measures that are well-established in TBI research for times early after injury, such as two weeks, until two months. Some of these methods have previously been used at later times after injury, up to one year, but by very few laboratories. The methods demonstrated here are a short neurological assessment to test reflexes, a Beam-Balance to test balance, a Beam-Walk to test balance and motor coordination, and a working memory version of the Morris water maze that can be sensitive to deficits in reference memory. Male rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid percussion injury (FPI) or sham injury. Rats can be tested on the short neurological assessment (neuroscore), the beam-balance, and the Beam-Walk multiple times, while testing on the water maze can only be done once. This difference is because rats can remember the task, thus confounding the results if repeated testing is attempted in the same animal. When testing from one to three days after injury, significant differences are detected in all three non-cognitive tasks. However, differences in the Beam-Walk task were not detectable at later time points (after 3 months). Deficits were detected at 3 months in the Beam-Balance and at 6 months in the neuroscore. Deficits in working memory were detected out to 12 months after injury, and a deficit in a reference memory first appeared at 12 months. Thus, standard behavioral tests can be useful measures of persistent behavioral deficits after FPI.

The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, ...