Table 1.

Description of the behavioral analysis.

Behavior	Description	Ethological interpretation	References
Locomotion	It can be registered automatically by video tracking softwares (e.g., Any-Maze®, Nodulus®). It is essential that the starting minute matches between the manual and automatic behavioral scoring. To see the details of the program configuration, refer to the corresponding tracking software's user manual. The locomotor activity can also be manually assessed by dividing the testing cage into two halves and then counting the number of lines crossed as the animal moves laterally.	Horizontal activity (i.e., locomotion) increases when animals face a novel situation or context serving exploratory and information-gathering purposes. Locomotion is a general indicator of psychomotor reactivity and an index of anxiety (the motivation to escape from unknown and unpredictable stimuli).	[8,12,19,28,30,31,36].
Rearing	Rearing consists of a bipedal posture (>45° from the floor), in which the animal extends its head upwards, executing a series of lateral movements using its vibrissae (whiskers) to sense the surroundings (by smelling and touching out). These exploratory movements with the head and vibrissae are referred to as sniffing. The animals may or may not lean against the walls to get up on their hind legs. Rearing should not be counted if the animals rise on its hind legs but keep its forepaws and head parallel to the ground, with an inclination of its torso <45°.	Rearing is considered a risk assessment (RA) behavior involved in evaluating possible threat sources during potentially dangerous or ambiguous situations. RA serves to predict the likelihood of threats and choose the best coping behaviors. Because uncertainty and ambiguity about possible threats naturally evoke rearing, it has been interpreted as an anxiety-like behavior.	[4,5,28,31].
Grooming	Grooming consists of a set of complex, self-directed movements aimed at the care of the body surface. It includes (1) hand rubbing, (2) face washing, (3) unilateral and bilateral strokes over the head and ears, (4) body licking, (5) anus-genital licking, (6) head and body scratching, and (7) tail licking. Micro grooming (<1s) and scratching events are sometimes excluded from the analysis. However, we strongly suggest that those sequences should not be neglected. The scratching events are counted as grooming only when they are chained with other sequences.	Self-grooming is a widespread behavior in the animal kingdom. Besides its cleaning purpose, grooming's biological functions involve communication, parasite control, thermoregulation, pain-relief, and many others. Grooming has also been associated with anxiety and stress and constitutes a critical behavioral marker in many models of neuropsychiatric diseases. Evidence suggests that different grooming subtypes have different interpretations and, therefore, may be analyzed separately. From our perspective, short, head directed grooming strokes are mainly related to on-going negative arousal, whereas the more complex sequences of grooming seem to be involved in emotional self-regulation. In that sense, we suggest that grooming would be part of an arousal-inhibiting system serving for homeostatic behavioral control. However, grooming interpretation in the context of behavioral sciences is still a vividly open discussion.	[1,8,12,[16], [17], [18], 21,22,25,28,[30], [31], [32],37,38].
USVs	Most rodent species, including laboratory rats and mice, emit USVs (i.e., >20 kHz) produced by a whistle mechanism through their larynx's constriction. In contrast to sounds generated by vocal fold vibration –which are readily audible to humans– these whistle-driven USVs are inaudible without specialized equipment. According to their average frequency, three groups of USVs have been identified so far: (1) 22 kHz (emitted by young and adult rats), (2) 40 kHz (emitted only by rat pups), and (3) 50 kHz USVs (emitted by young and adult rats). When the emission of 22 kHz USVs occurs, a particularly evident body posture is displayed (which does not occur for 50 kHz USVs). Stereotyped diaphragm contractions characterize this posture while the rat is relatively immobile.	USVs are a form of social-affective communication naturally emitted by rats under emotionally related situations. 50-kHz USVs are emitted during conspecific and hetero-specific play, reward anticipation, mating-related activities, and the exposure to different familiar or novel contexts. Animals exposed to a cage filled with fresh bedding material, such as the CT, also emit 50 kHz calls. In consequence, 50 kHz calls are related to positive affect and social coordination. Conversely, 22 kHz calls are emitted when the animals are exposed to acute stress events such as conspecific aggression, inescapable pain, or predator-related olfactory cues. Further, rats exposed to 22 kHz USVs show a substantial increase in defensive behaviors such as freezing. Therefore, 22 kHz USVs are considered as an index of a negative affective state.	[9], [10], [11], 14,24,27,[33], [34], [35]].