Adult Restoration of Shank3 Expression Rescues Selective Autistic-Like Phenotypes methods
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Tamoxifen Preparation and Feeding
Tamoxifen (Sigma #T5648) was dissolved in corn oil at 20 mg/ml through vortexing. Freshly prepared tamoxifen was protected from light by aluminum foil and kept for 2-3 days at room temperature. Animal feeding needles from Harvard Apparatus (cat #52-4025) were used for oral gavage. To avoid toxicity of tamoxifen, the following dosages were used for adult animals:
Tamoxifen Preparation and Feeding
Mice at 17-21 g body weight were fed 5 mg/day
Tamoxifen Preparation and Feeding
Mice at 22-25 g body weight were fed 6 mg/day
Tamoxifen Preparation and Feeding
Mice at 26-29 g body weight were fed 7 mg/day
Tamoxifen Preparation and Feeding
Mice at 30-35 g body weight were fed 8 mg/day
Full Methods
Animals were housed by genotype at a constant 23°C in a 12 h light/dark cycle (lights on at 07:00, lights dark at 19:00) with ad libitum food and water. Rescue treatment i.e. tamoxifen feeding was initiated on mice at 2-4.5 months. All electrophysiological and behavioral experiments were done at least 6 weeks after treatment in adult mice with the experimenter being blinded to the genotypes. Only age-matched male mice were used for behavioral assays. All experimental procedures were inspected and approved by the MIT Committee on Animal Care.
Open Field
An automated Omnitech Digiscan apparatus (AccuScan Instruments) was used to assess spontaneous locomotion as previously described. Anxiety-like behaviors were assessed by the following parameters: time spent rearing and frequency of rearing. Locomotion was evaluated by the total distance traveled. The first 30 minutes were evaluated for all parameters. Statistical analysis was done using one-way ANOVA with Bonferroni multiple comparison tests.
Zero Maze
An elevated zero maze was illuminated such that the open arm was lit by 60 lux, and the dark arm was lit by 10-20 lux. Animals were habituated with 10-20 lux for at least one hour before test. The animal was introduced into the closed arm and allowed to freely explore the maze for 5 minutes, which was videotaped. An observer blinded to the genotype performed analysis using an automated tracking software Noldus Ethovision. Anxiety-like behavior was assessed by the percentage of time spent by the animal in the open arm during the 5-min interval. Statistical analysis was done using one-way ANOVA with Bonferroni multiple comparison tests.
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Evidence quotes (8)
Tamoxifen (Sigma #T5648) was dissolved in corn oil at 20 mg/ml through vortexing. Freshly prepared tamoxifen was protected from light by aluminum foil and kept for 2-3 days at room temperature. Animal feeding needles from Harvard Apparatus (cat #52-4025) were used for oral gavage. To avoid toxicity of tamoxifen, the following dosages were used for adult animals:
Mice at 17-21 g body weight were fed 5 mg/day
Mice at 22-25 g body weight were fed 6 mg/day
Mice at 26-29 g body weight were fed 7 mg/day
Mice at 30-35 g body weight were fed 8 mg/day
Animals were housed by genotype at a constant 23°C in a 12 h light/dark cycle (lights on at 07:00, lights dark at 19:00) with ad libitum food and water. Rescue treatment i.e. tamoxifen feeding was initiated on mice at 2-4.5 months. All electrophysiological and behavioral experiments were done at least 6 weeks after treatment in adult mice with the experimenter being blinded to the genotypes. Only age-matched male mice were used for behavioral assays. All experimental procedures were inspected and approved by the MIT Committee on Animal Care.
An automated Omnitech Digiscan apparatus (AccuScan Instruments) was used to assess spontaneous locomotion as previously described. Anxiety-like behaviors were assessed by the following parameters: time spent rearing and frequency of rearing. Locomotion was evaluated by the total distance traveled. The first 30 minutes were evaluated for all parameters. Statistical analysis was done using one-way ANOVA with Bonferroni multiple comparison tests.
An elevated zero maze was illuminated such that the open arm was lit by 60 lux, and the dark arm was lit by 10-20 lux. Animals were habituated with 10-20 lux for at least one hour before test. The animal was introduced into the closed arm and allowed to freely explore the maze for 5 minutes, which was videotaped. An observer blinded to the genotype performed analysis using an automated tracking software Noldus Ethovision. Anxiety-like behavior was assessed by the percentage of time spent by the animal in the open arm during the 5-min interval. Statistical analysis was done using one-way ANOVA with Bonferroni multiple comparison tests.
Machine-readable layer
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