The aim of this procedure is to create a controlled environment which maximizes the occurrence of playful behavior and to analyze the organization of the resulting playful behavior. This is accomplished by first creating a natural setting for play fighting. This involves the behavior being conducted in the dark, as well as providing a test arena with bedding to provide a comfortable setting.
The second step is to habituate pairs of animals to the testing apparatus for three consecutive days for a duration of 30 minutes. Next, the animal should be isolated for 24 hours prior to play fighting testing. The final step is to test each pair in the play arena for 10 minutes.
Ultimately, use of these simple steps creates a context which facilitates play and can be used to reveal differences due to experimental manipulations. The main advantage of this technique compared to some other techniques such as recording, natural occurring behavior in a, in a litter is that large amounts of data can be collected in a short period of time, and the play partners can be standardized. Though this method can provide insight into playful interactions, it can also be applied to other systems such as other model species or strains.
Animals used for testing are weaned between postnatal days 21 to 23, and depending on the experimental design, animals can be housed together after weaning in groups of two to four in the required male and female combinations. Pairs that are housed together are also habituated together for three 30 minutes sessions directly prior to the onset of testing. To do this, the animals are transported into the darkened testing room.
Play habituation and testing occurs during an animal's light cycle. Although testing occurs in a dark room While they're testing the light phase or the dark phase of rat's daily cycle, there's our experience that play increases have tested under red light conditions compared to white light conditions. Although here we choose to test in complete darkness, The animals are placed in the play box for habitation.
The play box apparatus is a clear plexiglass box measuring 50 centimeters by 50 centimeters by 50 centimeters with one to two centimeters of standard corn cob bedding. After the first habituation session, subjects are removed from the play box and placed together in their home cage. Then the bedding is discarded and the play apparatus is thoroughly cleaned using the antiseptic vercon or an equivalent.
This is a superior agent for cleaning the rat odors from the previous pair as it does not leave the strong and persistent smell of alcohol wipes or sprays. New bedding is then added and the habitation session for the next pair commences. This process is repeated until all pairs have been habituated for three 30 minute sessions.
Following habituation pairs are individually isolated 24 hours prior to testing to maximize playful interactions. Brief periods of social isolation increases the motivation to engage in social play while not having a similar effect on other social behavior such as social investigation. Although any isolation has this effect, the impact of isolation on play reaches an asymptote around 24 hours.
On the day of testing, the habituated rats are transported to the darkened testing room. The separated pairs are then reunited when placed in the play box, ensure that the camcorder on the tripod is correctly placed at a 45 degree angle facing downwards towards the playbook, and that all four corners of the box are visible in the shot to obtain a full view of the apparatus, commence filming in night shot mode and test the pair for 10 minutes. While trials are five minutes can detect isolation induced changes in behavior, they're too short to detect subtle differences due to a ceiling effect.
By 15 minutes, the animals appear fatigued by the vigorous play and consequently, trials lasting 10 minutes appear to be a good compromise between the economy of protocol and also the ability to detect experimental effects. Once the 10 minutes have elapsed, remove the animals, then clean the play boxes before and test the next pair. This video shows an attacked rat employing evasive defense.
Here the defender can walk, run, leap or dodge away, and by doing so, it not only moves its nappe away from its attacker, but also ends up facing away from its attacker. As the rat on the far left approaches, the other's nappe, the recipient leaps forward and away from the attacker. A defensive action where the rat turns to face its attacker can occur in two types of AEs of rotation.
In the first, it rotates around the long axis of the body. So here's the nose, here's the body, and it rotates the body around. In this plane, in the other form, the rat turns to face, but around a vertical axis are keeping the body horizontal as it turns to face the other animal.
Now also in this longitudinal axis rotation, there is two distinct forms of this particular action. Complete rotation around the longitudinal axis involves the rat turning from a standing position to fully supine. As the rat on the left attacks the nape, the recipient moves the nap away by rolling around the longitudinal axis of its body and ends in a fully supine position.
Note that the roll onto the back is a continuous motion lasting three to four frames. Partial rotation around the longitudinal axis involves the rat turning its four quarters, but leaving one or both of its hind paws in contact with the ground. As the attacker in the front reaches for the nappe, the recipient rolls its four quarters away, but keeps its hind paws on the ground.
This orientation is maintained as the attacker continues to reach for the nappe. Horizontal rotation around the vertical axis requires the defender to move its rump away from its attacker while moving its head towards it. Here, as the attacker in the rear reaches for the nape, the recipient rotates around a vertical axis to face the attacker while keeping its back facing skywood.
Immediately following a vertical rotation defense or a partial longitudinal rotation defense, the defending animal can rear up towards its opponent and the attacking animal can simply rear up to face it so that they end up converging on this upright position, which tells us that irrespective of where the initial movements that began the defense were, they can converge on the same outcome. The possible confound of focusing on outcome rather than the initial movements is even more exaggerated, where we compare cases of complete rotation with partial rotation around the longitudinal axis. In this video, as the attacker reaches for the nape, the recipient rolls over onto its back, and so protects the nappe as shown above.
This view of this maneuver emphasizes the fluid and rapid defensive action, which lasts two to three frames. A partial rotation around the longitudinal can also sometimes lead the defending animal to end up lying on its back just like it does with a complete rotation. But how it gets there compared to a complete rotation is very different in a complete rotation as one animal attacks the nap or the other one, this one smoothly and continuously rolls over onto its back in a, in a case where it's performing a partial rotation, and so moving the nap away, this animal's attacking the nap and then not rotating any further than that.
But then the attacking animal can keep reaching around, pushing as he goes, leading the one on the that's defending to gradually roll further and further and further until it's on its back. So in this case, even though it lands on its back from a partial rotation, it's the, it's the pressing action and the continued attack of the attacking animal that leads it to that position, not because the animal actually selects to roll over onto its back. This video shows an example of the defender being forced into the supine position.
As the attacker reaches for the nape, the recipient rotates. Its four quarters moving. Its nape away from the attacker.
Then the attacker continues to press forward reaching for the nape, and the defender continues to maintain contact with at least one hind pour on the ground for 21 to 22 frames before ending in a fully supine position. As most studies are interested in the effect of particular manipulations on a subject score the behavior by the defensive tactic attempted by the subject rather than the eventual outcome. This judgment can be made by examining the movements of the defender within the first two to three frames of the commencement of a defensive action.
Playful attacks can be measured when the snout of one rat either contacts or comes within one to two centimeters of its partner's nape. As the rat on the right sniffs the other rat, it gradually moves snout contact from the rump up towards the shoulders. Once close to the nape, the recipient turns to face blocking further contact.
Note that the animal on the right never makes a targeted movement towards the nappe of the other rat, and so is not considered as an attack. As noted above, the defensive tactic of the recipient can be categorized in the first few frames following an attack. This video shows an extended play session.
The attacker reaches for the nape. The recipient rolls onto its back with the attack continuing to move towards the nape. Contact with the nape is repeatedly broken, but there is no interruption in the movements of attack and defense leading to the regaining of nape contact.
This video shows separate playful attacks as the rat on the left attacks the nape, the recipient rolls partially to its side. The attacker follows the nape, but then faces away breaking contact. It then leaps forward and turns to face again and launches a new attack.
In all cases, a new sequence of scoring begins with a new attack. Rats may counter attack once they have successfully defended themselves as seen here as the rat closest to the camera approaches the other. The recipient turns and attacks the nappe.
The defender then blocks the attack, regains its footing and notches its own attack. However, because CounterTack are highly correlated to the frequency of initiating attacks, the more complex phases of these interactions that need not be scored unless there is good reason to do so. When scoring play, there is a sequential analysis.
How many attacks are launched by the subjects? How many attacks are defended against and when defending? What is the likelihood of using the different tactics of defense?
Complete removal of the cortex can lead to adult rats that function relatively normally in laboratory environments. However, more subtle behavioral effects may be overlooked. The protocol outlined in this video was used to test whether players reduced in rats without a cortex.
The play fighting of juvenile and adult rats, corticated at birth was compared with sham treated controls. It can be seen that the number of pins, partial rotations, and complete rotations differed between intact and decorticate animals. Three adult rats, two males and a sterile female were housed together for a period of two weeks.
The animals were then tested impaired trials to determine which of the males in each triad was the subordinate one. The subordinates in some triads had their orbital frontal cortex removed, and in other triads, the subordinates were sham treated. This graph shows the change in the asymmetry demonstrated by the subordinate male rats when playing with the dominant colony male compared to the colony female.
The measure shows the percentage difference in using the complete rotation when interacting with these two types of partners. Lesions of the orbital frontal cortex abolish the subordinate's ability to play differentially with its cage mates, sham treated, subordinate males continue to show a strong asymmetry after the surgery Once mastered. This technique can be completed in one to two hours per film session.
If done properly While attempting this procedure, it's important to remember to be consistent with your coding methodology.
