Adult Restoration of Shank3 Expression Rescues Selective Autistic-Like Phenotypes methods
Aim. Evidence-backed execution summary for Adult Restoration of Shank3 Expression Rescues Selective Autistic-Like Phenotypes methods from Adult Restoration of Shank3 Expression Rescues Selective Autistic-Like Phenotypes.
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This experiment, in seven questions
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Full Methods
reagent used in the protocol.
- Use
- The Shank3 fx/fx targeting vector was designed by inverting the PDZ domain (exons 13 to 16) and flanking it with the FLEx cassette, which is composed of one pair of LoxP sites staggered with one pair of Lox2722 sites. Shank3 fx/fx conditional knock-in mice were generated by homologous recombination in R1 embryonic s...
Social Interaction
reagent used in the protocol.
- Use
- A modified version of the three-chamber social interaction assay was used as previously described,,. Only age-matched males were used for all tests. S129 males were used as stranger mice and were habituated to the test chamber for 3 sessions (20 minutes each) one or two days prior to the behavioral assay. On the...
Western blot
reagent used in the protocol.
- Use
- PSD and synaptosomal fractions of the striatum, cortex, and cerebellum were prepared as previously described. Purified fractions were separated on SDS-PAGE and quantified using Odyssey Licor. β-Actin and Tubulin were used as loading controls. Specific primary antibody for SAPAP3 was prepared as previously desc...
Dendritic Spine Analysis
reagent used in the protocol.
- Use
- Mice from WT (N=5), KO (N=4, 1 had to be euthanized due to lesion development), and TM (N=5) conditions at 6 to 12 months old, age-matched males were used. The pAAV-hSyn1-EGFP-P2A-EGFPf-WPRE-HGHpA construct was cloned and sent for commercial viral packaging by Upenn Viral Core with serotype 2/9. Farnesylated GFP (GF...
Electrophysiology slices
reagent used in the protocol.
- Use
- Acute striatal slices were prepared from 3-7 months old age-matched mice. Animals were anesthetized by avertin intraperitoneal injection (tribromoethanol, 20mg/ml, 0.5mg/g body weight) and transcardially perfused with ice-cold oxygenated NMDG-based cutting aCSF solution (mM): 92 N-methyl-D-glucamine (NMDG), 2....
Medial prefrontal cortex (mPFC)
reagent used in the protocol.
- Use
- Acute brain slices were prepared from 3-7 month-old mice as follows. Briefly, mice were deeply anesthetized by intra-peritoneal injection of avertin solution (20 mg/ml, 0.5 mg/g body weight) and then transcardially perfused with 20 ml of carbogenated (95% O 2, 5% CO 2 ) ice cold cutting solution with the comp...
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 tamoxife...
- Use
- 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 tamoxife...
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 minu...
- Use
- 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 minu...
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 vi...
- Use
- 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 vi...
Rotarod
Animals were placed on a rotarod apparatus (Med Associates) that accelerates 4-40 rpm for 5 minutes. Each animal was tested for three trials with 1-2 hours between trials in a single day. All trials were videotaped. Latency to fall was manually analyzed for each trial on Noldus Observer by an observer bl...
- Use
- Animals were placed on a rotarod apparatus (Med Associates) that accelerates 4-40 rpm for 5 minutes. Each animal was tested for three trials with 1-2 hours between trials in a single day. All trials were videotaped. Latency to fall was manually analyzed for each trial on Noldus Observer by an observer bl...
Social Interaction
A modified version of the three-chamber social interaction assay was used as previously described,,. Only age-matched males were used for all tests. S129 males were used as stranger mice and were habituated to the test chamber for 3 sessions (20 minutes each) one or two days prior to the behavioral assay. On the...
- Use
- A modified version of the three-chamber social interaction assay was used as previously described,,. Only age-matched males were used for all tests. S129 males were used as stranger mice and were habituated to the test chamber for 3 sessions (20 minutes each) one or two days prior to the behavioral assay. On the...
Dendritic Spine Analysis
Confocal images were taken with a 60X objective of the dorsal striatum. Spine count on intact neurons began 30-40 µm away from the soma and was extended for 10-60 µm from the origin. Spine density was analyzed automatically by Neuron Studio. All virus injections, imaging, and software analysis...
- Use
- Confocal images were taken with a 60X objective of the dorsal striatum. Spine count on intact neurons began 30-40 µm away from the soma and was extended for 10-60 µm from the origin. Spine density was analyzed automatically by Neuron Studio. All virus injections, imaging, and software analysis...
Electrophysiology slices
Acute striatal slices were prepared from 3-7 months old age-matched mice. Animals were anesthetized by avertin intraperitoneal injection (tribromoethanol, 20mg/ml, 0.5mg/g body weight) and transcardially perfused with ice-cold oxygenated NMDG-based cutting aCSF solution (mM): 92 N-methyl-D-glucamine (NMDG), 2....
- Use
- Acute striatal slices were prepared from 3-7 months old age-matched mice. Animals were anesthetized by avertin intraperitoneal injection (tribromoethanol, 20mg/ml, 0.5mg/g body weight) and transcardially perfused with ice-cold oxygenated NMDG-based cutting aCSF solution (mM): 92 N-methyl-D-glucamine (NMDG), 2....
Medial prefrontal cortex (mPFC)
Acute brain slices were prepared from 3-7 month-old mice as follows. Briefly, mice were deeply anesthetized by intra-peritoneal injection of avertin solution (20 mg/ml, 0.5 mg/g body weight) and then transcardially perfused with 20 ml of carbogenated (95% O 2, 5% CO 2 ) ice cold cutting solution with the comp...
- Use
- Acute brain slices were prepared from 3-7 month-old mice as follows. Briefly, mice were deeply anesthetized by intra-peritoneal injection of avertin solution (20 mg/ml, 0.5 mg/g body weight) and then transcardially perfused with 20 ml of carbogenated (95% O 2, 5% CO 2 ) ice cold cutting solution with the comp...
Zero Maze
Software used for acquisition, scoring, statistics, or reporting.
- Use
- 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 vi...
Dendritic Spine Analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Confocal images were taken with a 60X objective of the dorsal striatum. Spine count on intact neurons began 30-40 µm away from the soma and was extended for 10-60 µm from the origin. Spine density was analyzed automatically by Neuron Studio. All virus injections, imaging, and software analysis...
Statistical Analyses
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All statistical analyses were performed using Prism (GraphPad Software). All data sets were analyzed using D'Agostino-Pearson omnibus test and Shapiro-Wilk test for normality. Data sets with normal distributions were analyzed for significance using either unpaired Student's two-tailed t -test or ANOVA me...
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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.
Measurement outputs
What raw and processed outputs should exist?
The Shank3 fx/fx targeting vector was designed by inverting the PDZ domain (exons 13 to 16) and flanking it with the FLEx cassette, which is composed of one pair of LoxP sites s...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
An automated Omnitech Digiscan apparatus (AccuScan Instruments) was used to assess spontaneous locomotion as previously described. Anxiety-like behaviors were assessed by the f...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
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...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Animals were placed on a rotarod apparatus (Med Associates) that accelerates 4-40 rpm for 5 minutes. Each animal was tested for three trials with 1-2 hours between t...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
An automated Omnitech Digiscan apparatus (AccuScan Instruments) was used to assess spontaneous locomotion as previously described.
from paperScoring or quantification
Quantify the primary readouts for this experiment: The Shank3 fx/fx targeting vector was designed by inverting the PDZ domain (exons 13 to 16) and flanking it with the FLEx cassette, which is composed of one pair of LoxP sites s...; An automated Omnitech Digiscan apparatus (AccuScan Instruments) was used to assess spontaneous locomotion as previously described. Anxiety-like behaviors were assessed by the f...; 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...; Animals were placed on a rotarod apparatus (Med Associates) that accelerates 4-40 rpm for 5 minutes. Each animal was tested for three trials with 1-2 hours between t....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
An automated Omnitech Digiscan apparatus (AccuScan Instruments) was used to assess spontaneous locomotion as previously described. Anxiety-like behaviors were assessed by the f...; 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...; Animals were placed on a rotarod apparatus (Med Associates) that accelerates 4-40 rpm for 5 minutes. Each animal was tested for three trials with 1-2 hours between t...; Animals were individually placed into a new cage and allowed to habituate. Grooming behavior was videotaped for 2 hours from 19:00 to 21:00 with red light (2 lux). An observer b...
from paperReporting output
Report representative outputs alongside summary comparisons for The Shank3 fx/fx targeting vector was designed by inverting the PDZ domain (exons 13 to 16) and flanking it with the FLEx cassette, which is composed of one pair of LoxP sites s..., An automated Omnitech Digiscan apparatus (AccuScan Instruments) was used to assess spontaneous locomotion as previously described. Anxiety-like behaviors were assessed by the f..., 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..., Animals were placed on a rotarod apparatus (Med Associates) that accelerates 4-40 rpm for 5 minutes. Each animal was tested for three trials with 1-2 hours between t....
inferred from protocolStructured statistical methods
An automated Omnitech Digiscan apparatus (AccuScan Instruments) was used to assess spontaneous locomotion as previously described. Anxiety-like behaviors were assessed by the f...; 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...; Animals were placed on a rotarod apparatus (Med Associates) that accelerates 4-40 rpm for 5 minutes. Each animal was tested for three trials with 1-2 hours between t...; Animals were individually placed into a new cage and allowed to habituate. Grooming behavior was videotaped for 2 hours from 19:00 to 21:00 with red light (2 lux). An observer b...
source structuredSource and audit
What supports the facts on this page?
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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