A Laboratory Method to Measure Contagious Yawning in Rats

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 communication^2,3. 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 afternoon^8,9.

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 vertebrates^{11,12,13,14,15,16}, 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 debate^20,21, 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'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 video^12,13; hence, contagion can only occur through visual cues. Other investigations have assessed yawn contagion in more natural conditions using groups of animals^14,15. 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's use of a given sense, then infer the role of that sense in the relevant behavior^2,3,18,22. Fortunately, there are other methods in which only physical barriers are used to either allow or impede communication between animals^23,24,25, 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 yawning^25,28. Dr. Stephen E. G. Lea (personal communication, 2015) helped us numerically adapt a method previously employed by primatologists^13,14 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'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.

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Protokół

The experimental protocols and animal husbandry were conducted in accordance with institutional guidelines.

1. Materials

1. 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.
2. 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).
3. 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.
   1. 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.
4. 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.
5. 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.

2. Procedure

1. 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.
2. 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.
3. 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 http://cran.r-project.org/) as follows [after the prompt (>)]:
   > sample(4,4)
   [1] 4 3 2 1
   1. 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.
   2. 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.
4. 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.
   1. 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:
      >sample(8,8)
      [1] ​8 7 4 6 5 1 2 3
   2. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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.
12. 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.
13. 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.
    1. 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).
    2. 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.

3. Data processing

1. 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 or NA (see below).
   NOTE: Use the general guide provided as a supplementary document to fully understand the following steps to process the data, measure contagious yawning, and obtain contagious yawning curves.
   1. 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).
   2. If yawning was recorded to the nearest decimal of a minute, type in the sequence top down and use "NA" 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.
2. Initiate R. Import the data from the file in which they were previously located.
3. Save the program codes with the extension ".R" (provided as supplementary material), 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.
   1. 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's workspace.
4. 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.
   1. 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.
   2. 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).

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Wyniki

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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Dyskusje

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...

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Materiały

Name	Company	Catalog Number	Comments
Acrylic dividers	Handcrafted	Not available	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.
An R-based program	Benemérita Universidad Autónoma de Puebla	Not available	This is the program used to assess yawn contagion in rats. See the main text for information about the way the program is used.
Data sheets	The user can elaborate them	Not available	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.
Desktop computer	Any maker	Not available	Make sure the computer has a video card capable of conveniently processing the video recordings of yawning behaviour.
Digital camcorders	Any maker	Not available	They will be used to video record yawning behaviour of each pair of rats; there will be 2 pairs of rats per experimental session.
Flash drive	Any maker	Not available	Each experimental session will last 60 min, and so you will require sufficient memory to store the video recording.
Glass cages	Handcrafted	Not available	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.
Markers	Sharpie or any other maker	Not available	Permanent markers to number the rats. See the main text to see one way of using painting symbols on the rat's tail.
Pencils	Any maker	Not available	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.
R software	R Development Core Team	Not available	Download R at: http://cran.r-project.org/
Rail-like wooden bars	Handcrafted	Not available	They will be fixed in the middle of the rectangular wooden sheet  forming a track, where a second wooden sheet is placed. See the main text for additional instructions for construction.
Rectangular table	Any maker	Not available	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.
Sprague-Dawley male rats	Any local supplier of laboratory animals	Not available	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.
Spreadsheet software	Microsoft	Not available	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.
Square filter papers	Any maker	Not available	They are used for covering the cage's bottom.
Tripods	Any maker	Not available	They will be used for fixing the camcorders in front of each pair of observation cages.
Wooden Inverted T-shaped table	Handcrafted	Not available	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.