Mice
Mouse work was performed in accordance with the guidelines of the animal ethics committee of the University of Iowa. Mice were group-housed in a colony maintained with a standard 12 h light/dark cycle and given food and water ad libitum. Experiments were performed on age-matched mice of both sexes as indicated in bar graphs. Experiments were conducted according to the Guide for the Care and Use of Laboratory Animals, as adopted by the National Institutes of Health, and with approval of the University of Iowa AAALC-accredited Institutional Animal Care and Use Committee.
The Sacs-/- mice were a kind gift of Bernard Brais, McGill (3). The AKAP1 -/- mouse line was kindly provided by Dr. Stanley McKnight at University of Washington (29), and Bβ2-/-mice were generated at the U. Iowa Mouse KO Core Facility (11). We generated mice with heterozygous or homozygous deletion of PP2A/Bβ2 or Akap1 that were either wild-type or null at the Sacs locus. Mice were in the C57BL/6J background and were backcrossed to C57BL/6J mice imported from the Jackson Laboratory (Bar Harbor, ME) every 6-10 generations to prevent genetic drift. Mice of all genotypes were born in expected Mendelian ratios and were fertile, except for Akap1-/- females who are infertile (29). All mice achieved a normal lifespan, with many individuals surviving beyond two years. However, Sacs-/- mice with or without deletion of Bβ2 or Akap1 displayed progressive gait abnormality as documented (3).
Behavioral Testing
General. 5-7 days of habituation and handling was done prior to behavioral assessment. Mice were allowed to acclimatize to the testing environment for 30 minutes prior to the starting of the experiment trials on the day of testing. All genotypes were tested on the same day in a randomized order with experimenters blinded to genotype and sex.
Accelerating rotarod. Mice were placed on a rotating rod (IITC Life Woodland Hills, CA) with gradual speed increase from 4 to 40 rpm over 5 minutes, in which the latency to fall was recorded. Testing consisted of four trials on each day for three consecutive days, with 5-10 minutes between each trial. The average of four daily trials was recorded for each mouse.
Balance beam. The balance beam apparatus was acquired from MazeEngineers Inc (Skokie, IL). The balance beam test was performed as previously described (30). A 12 mm-wide beam was used in our tests. Mice were placed at one end of the beam and the latency for crossing the beam to the other end was recorded by MediaRecorder software (Noldus). Mice were trained for three times in each day for two consecutive days and tested for three times on day 3. The best performance (minimum latency to cross beam) was recorded on the testing day.
Wire hang test. The test began with the mice placed on an elevated wire cage top, which was then inverted and suspended above the home cage. The time it took for the animal to fall was recorded. This test was conducted two times on one day, with 5-10 minutes between each trial. The best performance (longer hang time) was recorded.
Contextual Fear Conditioning. The testing apparatus for 24-hour contextual recall was acquired from CleverSys Inc (Reston, VA). Conditioning was assessed using FreezeScan V.2 software, which measures the "freezing" behavior of the mouse. On the training day, the animals underwent a 3-minute trial, with an electric shock of 1.5 mA delivered at 2.5 min and lasting for two seconds. On the testing day, the animals were returned to the exact same environment and underwent a 5-minute trial without any shocks. Data were presented as percentage of time freezing on the testing day.
Statistical Analysis
Data were obtained and analyzed with experimenters blinded to genotype and sex. Statistics were analyzed, and plots were generated using GraphPad Prism software (version 10.2). All data were first analyzed by D’Agostino-Pearson test to determine the normality and then analyzed by two-way ANOVA with Dunnett’s multiple comparison tests. Motor learning was analyzed by linear regression. The false positive rate (a) was set at 0.05.