Behavioral tests for phenotype characterization
A group of 25 WT and 12 Plcb1+/- mice underwent different behavioral tests for phenotype characterization for 10 days, as described in Figure 1:
Short-term memory. To measure short-term memory, the novel object recognition (NOR) test was used. This test was performed in a V-maze apparatus (40 cm per side, Panlab, Spain) as previously described by us and others (Bura et al., 2010; Planaguma et al., 2015). On day 1 mice were habituated for 9 minutes in the V-maze. On day 2, mice were put back into the V-maze for 9 minutes; two identical objects were presented, and the time the mice spent exploring each object was recorded. After a retention phase of 3 hours, the mice were placed for another 9 minutes into the V-maze; in each paradigm, one of the familiar objects was replaced with a novel object and the total time spent exploring each object (novel and familiar) was registered. Object exploration was defined as the orientation of the nose to the object at a distance of less than 2 cm. A discrimination index was calculated as the difference in the time spent exploring the novel and the time spent exploring the familiar object divided by the total time exploring both objects. A higher discrimination index is considered to reflect more excellent memory retention for the familiar object.
Locomotor activity. Locomotor activity was assessed in locomotor activity boxes (9×20×11 cm; Imetronic, Passac, France), equipped with 2 rows of photocell detectors, and placed in a low-luminosity environment (20–25 lux), as previously described (Martin-Garcia et al., 2013). The locomotor activity was recorded for 2 hours, and 2 variables were measured: horizontal activity as horizontal displacement and vertical activity as vertical exploration/elevation.
Elevated plus maze test. This test was performed as previously reported (Planaguma et al., 2015). The test measures the conflict between the natural tendency of mice to avoid an illuminated and elevated surface, and the natural tendency to explore new environments. It consisted of a black plastic cross with arms 40 cm long and 6 cm wide placed 50 cm above the floor. Two opposite arms were surrounded by walls (15 cm high, closed arms, 10 lux), while the two other arms were devoid of such walls (open arms, 200 lux). A central platform connected the four arms. At the start of the session, the mouse was placed at the end of a closed arm facing the wall. During the 5-minutes trial, the number of entries and the time spent in each arm were recorded. Anxiety was assessed as both the time spent avoiding the open arms and the number of entries into them.
Motor coordination. Rota rod apparatus was used to assess motor coordination. Precisely, this test measures the ability of the mouse to remain on a rotating rod in which the speed of rotation is gradually increased and evaluate general motor coordination. The device is a round drum (5 cm diameter) suspended between 2 plexiglass walls. The drum is suspended at 24 cm from a soft mat covered tabletop. The speed of rotation is controlled by an electric engine with a digital revolution per min (rpm) display. The test consisted in five trial sessions. Each trial started with the mouse being placed on the rotating rod at 4.0 rpm, and then every 3 s the speed increased by 1.0 rpm until 20.0 rpm. The trial terminated when the mouse fell from the rod or after 90 s, whichever occurred first. There was a 10 s interval between trials. The average maximum rota rod speed and time to fall was calculated for each mouse (Martin-Garcia et al., 2013).