During the training session, mice were placed in the conditioning chamber (TSE Systems, Inc.), with a striped pattern, for 2.5 min, before receiving a 0.7 mA foot shock for 2 s. Prior to the training session, mice were handled for 1–2 min per day for 3 days. The assay was split into a training and retention session. The Contextual Fear Conditioning Assay was performed as previously described 81. The ANY-MAZE software was used to record and analyze immobility (Stoelting Co.). For the last 4 min, mice were videotaped and the immobility time was recorded. During the 6-min assay, the first 2 min of activity was discarded. The forced swim assay was performed as previously described 84. Tests were video recorded and analyzed by the ANY-MAZE software (Stoelting Co.). The relative exploration time was recorded and expressed by a D.I.: Equation (1). During both the training and testing phases, the duration and number of times the mice interact with the familiar and novel objects were recorded individually. Twenty-four hours later, one of the familiar objects was replaced with a novel object and the animals were given 5 min to explore the chamber.
During the training phase, mice were placed in the experimental apparatus, and were given 10 min to explore and examine two identical objects. Prior to training, the mice were handled for 1–2 min a day for 3 days and were given 10 min a day for 3 days inside the empty experimental apparatus to habituate to the environment. The novel object recognition (NOR) assay consists of training and testing phases 81. The time for the animal to decide on a side arm (decision latency) was also recorded. The alternation percentage was calculated as 100 x (number of alternations/7).
A total of eight trials were completed, allowing for a total of seven total possible alternations. After a choice (all four paws in the chosen arm), the sliding door was closed behind the animal, allowing the mouse to explore the chosen arm for 30 s, before being manually returned to the start area for the next trial. After the acclimation period, the sliding door was removed, and the mice can choose either the left or right-side arm. The mice were placed in the starting area behind a sliding door, blocking the main stem and side arms and were given a 30 s acclimation period before the start of each trial. The T-maze has three designated parts: the main stem, the side arms, and the starting area (AccuScan Instruments, Inc.) 82. The test was video recorded and analyzed by the ANY-MAZE software (Stoelting Co.).
The relative exploration time was recorded and expressed by a discrimination index. The duration of direct contact between the subject mouse and both the empty cup and the cup with the mouse was measured. The dividing doors were then removed to allow the mouse to freely travel between the three chambers for 10 min. The subject mouse was placed in the middle chamber and was given 5 min to explore with the dividing doors closed. In one of the cups on either side, a control mouse (an unfamiliar mouse of the same strain, gender, and age with no prior contact with the subject mouse) was placed. Two wire mesh cups were placed in the middle of both the right and left chamber (one on each side). The social interaction test was performed using the three-chambered apparatus 82. The animals were scored based on time spent in the closed and open arms and the number of entries to the closed and open arms. The behavior was recorded and scored by two independent observers blind to the animal treatments.
#ANY MAZE SOFTWARE COPY LICENCE PLUS#
During the test, the mice were placed in the center of the plus facing an open arm and were given 5 min to explore. Animals were given a 30-min period to habituate to the room before being tested. A standard elevated plus-maze, made of grey Plexiglas, was placed in a sound-proof observation room with controlled light (200 Lux) on the central platform of the maze 83.
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