4.5. Behavioral assays

All behavioral assays were conducted in accordance with previously published procedures from using conditions that were demonstrated to detect differences in anxiety-like and affective behaviors^9,43,50,52. The order in which the tests were conducted was as follows: 1) EPM, 2) open field, 3) NSF, 5) forced swim, 6) fear conditioning. Animals were given at least 48 hours to recover after the EPM and open field tests and at least 7 days after the NSF and forced swim tests before further behavioral testing was conducted. A subset of animals was tested in the following order: 1) EPM, 2) looming disc, 3) restraint plus EZM and 4) fear conditioning.

This test was conducted according to previously published results which were found to detect anxiogenic effects of fluoxetine in mice⁹. Mice were placed in the center of an elevated plus maze, which consisted of one open arm (35 cm × 5 cm) crossed at a right angle by an enclosed arms (35 cm × 5 cm) with walls that are 15 cm in height. The entire arena is raised at a height of 60 cm from the floor. The mice were allowed to freely explore during a 5 min session. Light levels in the open arms were at ~ 25 lux. Time spent in the open arm, latency to the open arm, open arm probability, and total distance moved were measured.

Mice were placed into the corner of a white Plexiglas open field arena (50 × 50 × 50 cm) and allowed to freely explore for 30 min. Time spent in the center, latency to the center, the number of center entries, and the total distance moved were measured. The center of the open field was defined as the central 25% of the arena. The open field test took place either under high light conditions (300 lux) or low light (25 lux) conditions.

Forty-eight hours before testing, mice were provided with access to a single piece of Froot Loops cereal (Kellogg’s) in their home cage. Twenty-four hours before testing, home cage chow was removed. Mice were weighed both before food deprivation and before testing to assess body weight loss. Water remained available ad libitum. Beginning at least one hour before testing, mice were transferred to a new clean cage so that they were singly housed for the testing session. During the testing session mice were placed into a brightly lit arena (50 × 50 × 50 cm) that contained a single Froot Loop on top of a piece of circular filter paper. Mice were monitored by a live observer and the latency for the mouse to begin eating the pellet was measured. Following the initiation of feeding, mice were removed from the arena and placed back into their home cages. Mice were then provided with 10 min of access to a pre-weighed amount of Froot Loops for a post-test feeding session. After this 10 min post-test, the remaining Froot Loops were weighed, and mice were returned to ad libitum home cage chow. Mice were returned to group housing at the end of this session.

Mice were placed inside of a 50 ml conical tube with air holes throughout the body of the tube which was taped down to a lab bench for 30 minutes. Immediately afterwards, they were placed in the elevated zero maze (EZM) for behavioral testing. The EZM has a diameter of 60 cm with a 5 cm wide circular corridor that has 16 cm high walls on opposite sides. Time spent in the open areas of the maze is taken as an inverse measure of anxiety-like behavior.

Forced swim procedures were carried out as described previously⁴³. Briefly, mice were placed in a clear, plexiglass container that was filled with 24°C to a height of 15 cm for 6 min. Immobility during the last 4 minutes was hand-scored using Ethovision XT 10 and used as a measure of behavioral despair. Animals exhibiting behavioral despair will spend a higher percentage of their time in an immobile state⁶².

A two-day protocol was used to assess contextual fear recall. On the first day, mice were placed into a fear conditioning chamber (Med Associates) containing a grid floor. Prior to the session, the chamber was cleaned with a scented paper towel (19.5% ethanol, 79.5% H2O, 1% vanilla). After a 3 min baseline period during which mice were allowed to explore freely, mice were exposed to a 30 s tone (3 kHz, 80 dB) that co-terminated with a 2 s scrambled foot shock (0.6 mA). A total of 5 tone-shock pairings were delivered with a random inter-tone interval (ITI) of 60–120 s. Following delivery of the last foot shock, mice remained in the conditioning chamber for a 2-min consolidation period. The following day, mice were returned to the same training chamber for 10 min and allowed to freely explore. Freezing behavior was scored at every 5 second interval and averaged for the first 3 minutes. For each session, cameras were mounted overhead for recording freezing behavior, which was later hand-scored every 5s by a trained observer blinded to experimental treatment. Freezing was defined as a lack of movement except as required for respiration.

The looming disc assay was performed as described previously.^50,52 Mice were placed inside of an open top plexiglass box with white matte flooring (40 × 40 × 30 cm) and a dark shelter nest in far left corner. The nest was in the shape of a triangular prism (20 × 12 cm). An LED monitor with a gray screen was placed on top of the arena, which would display an expanding black disc when triggered. The floor and three walls were covered with a matte coating to prevent reflections of the stimulus. Infrared LEDs were placed around the outer edges of the area for video recording. Htr1a^fl/fl mice aged 8–12 weeks were group-housed and maintained on a regular light/dark cycle. On the day of looming disc test, each mouse was allowed to freely explore the looming box for 10 min. At the end of the habituation phase, an experimenter would trigger the looming disc when the mouse was at the center of the arena. Flight latency (ms) and velocity to escape into the nest were measured by trained observer blinded to the experimental conditions.