Our protocol is significant because it provides an easy way to investigate which senses may be involved in contagious yawning in rats. The use of a non-invasive procedure and the availability of the application software, which speeds the calculation for measuring contagious yawning, are the main advantages of this technique. Individuals who have never performed this technique shouldn't face major difficulties in the implementation, provided they methodically follow each step and seek professional advice when constructing the observation case.
Visual demonstration of this method aids in understanding the spatial arrangement of the observation cages during the experiment. Helping to avoid errors in designation of the treatments. Demonstrating that procedure will be Evelyn Beristain-Castillo, a postdoc from my lab.
To set up a test session, transfer four rats into the observation room in individual cages. While the animals are acclimating, place an inverted T-shaped table on a larger rectangular table under a ceiling lamp that sufficiently lights the room. Place 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 a divider between each pair of cages, and strategically place two digital camcorders, such that each records the yawning behavior of each pair of rats. After confirming that the camcorders are securely fixed to tripods and correctly oriented to the observation cages, connect the camcorders to a desktop computer to simultaneously monitor the behavior of the rats. When the animals have acclimated for 15 minutes, place them in the observation cages according to the previously determined allocation, and set the automatic focus to off on each camcorder.
Start the video recording and a stopwatch at the same time, stopping the video recording at the end of the 60 minute observation period. When the observation is over, return the rats to their home cages in the animal facility with access to food and water. Then thoroughly clean the observation cages with a non-toxic detergent before preparing the experimental set up to perform the subsequent test sessions for the day.
After the last session, play back each video on a computer screen using any standard playback system for an observer. Have the observer first view each video to observe and record the yawning behavior onto data sheets once at normal speed, followed by additional viewing at slower speeds to make it easier to observe and measure the yawning. To score the yawning, use vertical lines to represent the occurrence of a yawn with the minute written as a superscript.
When all of the yawns have been scored, transcribe the temporal sequence of yawns in each rat to a spreadsheet. If the yawning was recorded to the nearest minute, enter the number of yawns at each relevant minute and use zero to fill in the cells when no yawn occurred in a given minute. Then save the worksheet as a text file.
If the yawning was recorded to the nearest decimal of a minute, type in the sequence top down. Use NA to fill in the empty spaces of the rat columns in which the number of yawns in the rows is lower than that of the column. With the maximum number of rows of yawns recorded for a rat.
Then save the worksheet as a text file. When all of the data have been entered, import the text file into R.Save the program codes with the extension code R and download the specific program code depending on whether the yawns were recorded as integers or fractional numbers. Then run the program for each pair of rats and the desired number of time frames.
At the end of the run, run the program again using a random distribution of the number of yawns from each rat. To create yawn contagion curves, use the data previously saved in the spreadsheet to subtract the non-contagion rates from the contagion rates for each pair of rats, the time window, and the test situation, separating the analyses of the observed data and artificial data. In this representative experiment, the yawn contagion between unfamiliar male rats was measured over a range of time windows that varied from one to 10 minutes.
These yawn contagion curves indicate that only olfactory communication between rats in cages with a perforated opaque divider exhibited yawn contagion. Which is evident from the time window four and onward, as the confidence interval of the averages does not overlap with the confidence interval of the randomly allocated number of yawns over the 60 minute period. The band indicated, as expected, that a random allocation of the number of yawns in each rat in the observation period, produced yawn rates that oscillated at approximately zero without demonstrating any apparent pattern.
In this representative experiment with familiar male rats, none of the four test situations stimulated yawn contagion because their confidence intervals overlapped with the randomly generated confidence interval band. Further, there were no differences among the four test situations even though the overall increase in yawn contagion over the 10 time windows different from a zero slope. Remember to keep track of the familiar and unfamiliar identities of the rats for a correct interpretation of the results, and to clean the observation cages between experiments.
A pharmacological procedure may be used to block auditory communication, and to completely separate the hearing effects on contagious yawning from the effects from the other senses. This technique has paved the way for comparing contagious yawning across the species both in terms of the senses involved, and the strength of the response by comparing the contagious yawning curves. Remember to carefully handle the observation cages because they can accidentally break and cause injuries.
