Inhibition of mTOR by Rapamycin Abolishes Cognitive Deficits and Reduces Amyloid-β Levels in a Mouse Model of Alzheimer's Disease methods
Aim. Evidence-backed execution summary for Inhibition of mTOR by Rapamycin Abolishes Cognitive Deficits and Reduces Amyloid-β Levels in a Mouse Model of Alzheimer's Disease methods from Inhibition of mTOR by Rapamycin Abolishes Cognitive Deficits and Reduces Amyloid-β Levels in a Mouse Model of Alzheimer's Disease.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Western blotting and Aβ determinations
reagent used in the protocol.
- Use
- Mice were euthanized by isoflurane overdose followed by cervical dislocation. Hemibrains were flash frozen. One hemibrain was homogenized in liquid N 2 while the other was used in immunohistochemical determinations (5-6 per group) and for hippocampal dissections (5-6 per group). Half brains were microdis...
Behavioral testing
The Morris water maze (MWM),, was used to test spatial memory. All animals showed no deficiencies in swimming abilities, directional swimming or climbing onto a cued platform during pre-training and had no sensorimotor deficits as determined with a battery of neurobehavioral tasks performed prior to testing. All g...
- Use
- The Morris water maze (MWM),, was used to test spatial memory. All animals showed no deficiencies in swimming abilities, directional swimming or climbing onto a cued platform during pre-training and had no sensorimotor deficits as determined with a battery of neurobehavioral tasks performed prior to testing. All g...
Western blotting and Aβ determinations
Mice were euthanized by isoflurane overdose followed by cervical dislocation. Hemibrains were flash frozen. One hemibrain was homogenized in liquid N 2 while the other was used in immunohistochemical determinations (5-6 per group) and for hippocampal dissections (5-6 per group). Half brains were microdis...
- Use
- Mice were euthanized by isoflurane overdose followed by cervical dislocation. Hemibrains were flash frozen. One hemibrain was homogenized in liquid N 2 while the other was used in immunohistochemical determinations (5-6 per group) and for hippocampal dissections (5-6 per group). Half brains were microdis...
Immunohistochemistry
Ten-micrometer coronal cryosections from snap-frozen brains were post-fixed in 4% paraformaldehyde and stained with LC3-specific antibodies (10 µg/ml, Nous, Littleton, CO) followed by AlexaFluor488-conjugated donkey anti-rabbit IgG (1∶500, Molecular Probes, Invitrogen, CA), and imaged with a epifluorescen...
- Use
- Ten-micrometer coronal cryosections from snap-frozen brains were post-fixed in 4% paraformaldehyde and stained with LC3-specific antibodies (10 µg/ml, Nous, Littleton, CO) followed by AlexaFluor488-conjugated donkey anti-rabbit IgG (1∶500, Molecular Probes, Invitrogen, CA), and imaged with a epifluorescen...
Statistical analyses
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- Statistical analyses were performed using GraphPad Prism (GraphPad, San Diego, CA) and StatView. In two-variable experiments, two-way ANOVA followed by Bonferroni's post-hoc tests were used to evaluate the significance of differences between group means. When analyzing one-variable experiments with more than 2 group...
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Methods
The derivation and characterization of PDAPP [hAPP(J20)] mice has been described elsewhere,,. PDAPP mice were maintained by heterozygous crosses with C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME). PDAPP mice were heterozygous with respect to the transgene. Non-transgenic littermates were used as controls. Rapamycin administration and behavioral experiments involving PDAPP mice were conducted at the Buck Institute for Age Research, Novato, CA. Experimental groups were: control-fed non-Tg, n = 10; rapamycin-fed non-Tg, n = 10; control-fed Tg, n = 12; rapamycin-fed Tg, n = 12, all animals were males and 7 month-old at the time of testing. Rapamycin was administered for 13 weeks starting at 4 months of age.
Rapamycin treatment
Mice were fed chow containing either microencapsulated rapamycin at 2.24 mg/kg or a control diet as described by Harrison et al.. Rapamycin was used at 14 mg per kg food (verified by HPLC). On the assumption that the average mouse weighs 30 gm and consumes 5 gm of food/day, this dose supplied 2.24 mg rapamycin per kg body weight/day. All mice were given ad libitum access to rapamycin or control food and water for the duration of the experiment. Body weights and food intake were measured weekly. Food consumption remained constant for both control- and rapamycin-fed groups during treatment (no significant effect of week # on food consumption by two-way ANOVA, P = 0.108). Food consumption was higher for rapamycin-fed animals (by an average of 2.12±0.22 g/mouse/week at all times during the experiment ( P <0.001, two-way ANOVA). This may be a result of the inhibition of...
Behavioral testing
The Morris water maze (MWM),, was used to test spatial memory. All animals showed no deficiencies in swimming abilities, directional swimming or climbing onto a cued platform during pre-training and had no sensorimotor deficits as determined with a battery of neurobehavioral tasks performed prior to testing. All groups were assessed for swimming ability 2 days before testing. The procedure described by Morris et al. was followed as described,. Briefly, transgenic and non-transgenic PDAPP mice were given a series of 6 trials, 1 hour apart in a light-colored tank filled with opaque water whitened by the addition of non-toxic paint at a temperature of 24.0±1.0°C. In the visible portion of the protocol, animals were trained to find a 12 × 12-cm submerged platform (1 cm below water surface) marked with a colored pole that served as a landmark placed in different quadrants...
Western blotting and Aβ determinations
Mice were euthanized by isoflurane overdose followed by cervical dislocation. Hemibrains were flash frozen. One hemibrain was homogenized in liquid N 2 while the other was used in immunohistochemical determinations (5-6 per group) and for hippocampal dissections (5-6 per group). Half brains were microdissected to isolate the hippocampus by peeling away the cortex from the underlying hippocampus and releasing the hippocampus from the surrounding tissue, in particular the fimbria, by using fine surgical tweezers (Fine Science Tools) and then lifting toward the midline. The hippocampus separates easily but does include some adjacent white matter, which is then carefully tweezed off. We also obtained hippocampal tissue from at least 12 × 10 µm unfixed frozen sections mounted on glass slides. All but the hippocampal area was removed using a scalpel under a SZ60 Olympus...
Measurement outputs
What raw and processed outputs should exist?
The Morris water maze (MWM),, was used to test spatial memory. All animals showed no deficiencies in swimming abilities, directional swimming or climbing onto a cued platform...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Mice were euthanized by isoflurane overdose followed by cervical dislocation. Hemibrains were flash frozen. One hemibrain was homogenized in liquid N 2 while the other was used...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
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
Mice were fed chow containing either microencapsulated rapamycin at 2.24 mg/kg or a control diet as described by Harrison et al.
from paperScoring or quantification
Quantify the primary readouts for this experiment: The Morris water maze (MWM),, was used to test spatial memory. All animals showed no deficiencies in swimming abilities, directional swimming or climbing onto a cued platform...; Mice were euthanized by isoflurane overdose followed by cervical dislocation. Hemibrains were flash frozen. One hemibrain was homogenized in liquid N 2 while the other was used....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Mice were fed chow containing either microencapsulated rapamycin at 2.24 mg/kg or a control diet as described by Harrison et al.. Rapamycin was used at 14 mg per kg food (verif...; Statistical analyses were performed using GraphPad Prism (GraphPad, San Diego, CA) and StatView. In two-variable experiments, two-way ANOVA followed by Bonferroni's post-hoc tes...
from paperReporting output
Report representative outputs alongside summary comparisons for The Morris water maze (MWM),, was used to test spatial memory. All animals showed no deficiencies in swimming abilities, directional swimming or climbing onto a cued platform..., Mice were euthanized by isoflurane overdose followed by cervical dislocation. Hemibrains were flash frozen. One hemibrain was homogenized in liquid N 2 while the other was used....
inferred from protocolStructured statistical methods
Mice were fed chow containing either microencapsulated rapamycin at 2.24 mg/kg or a control diet as described by Harrison et al.. Rapamycin was used at 14 mg per kg food (verif...; Statistical analyses were performed using GraphPad Prism (GraphPad, San Diego, CA) and StatView. In two-variable experiments, two-way ANOVA followed by Bonferroni's post-hoc tes...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (4)
The derivation and characterization of PDAPP [hAPP(J20)] mice has been described elsewhere,,. PDAPP mice were maintained by heterozygous crosses with C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME). PDAPP mice were heterozygous with respect to the transgene. Non-transgenic littermates were used as controls. Rapamycin administration and behavioral experiments involving PDAPP mice were conducted at the Buck Institute for Age Research, Novato, CA. Experimental groups were: control-fed non-Tg, n = 10; rapamycin-fed non-Tg, n = 10; control-fed Tg, n = 12; rapamycin-fed Tg, n = 12, all animals were males and 7 month-old at the time of testing. Rapamycin was administered for 13 weeks starting at 4 months of age.
Mice were fed chow containing either microencapsulated rapamycin at 2.24 mg/kg or a control diet as described by Harrison et al.. Rapamycin was used at 14 mg per kg food (verified by HPLC). On the assumption that the average mouse weighs 30 gm and consumes 5 gm of food/day, this dose supplied 2.24 mg rapamycin per kg body weight/day. All mice were given ad libitum access to rapamycin or control food and water for the duration of the experiment. Body weights and food intake were measured weekly. Food consumption remained constant for both control- and rapamycin-fed groups during treatment (no significant effect of week # on food consumption by two-way ANOVA, P = 0.108). Food consumption was higher for rapamycin-fed animals (by an average of 2.12±0.22 g/mouse/week at all times during the experiment ( P <0.001, two-way ANOVA). This may be a result of the inhibition of the mTOR pathway, which is expected to mimic the unfed state by decreasing mTOR activity. Littermates (transgenic and non-transgenic mice) were housed together, thus we could not distinguish effects of genotype on food consumption. In spite of the differences in food consumption, overall body weigh...
The Morris water maze (MWM),, was used to test spatial memory. All animals showed no deficiencies in swimming abilities, directional swimming or climbing onto a cued platform during pre-training and had no sensorimotor deficits as determined with a battery of neurobehavioral tasks performed prior to testing. All groups were assessed for swimming ability 2 days before testing. The procedure described by Morris et al. was followed as described,. Briefly, transgenic and non-transgenic PDAPP mice were given a series of 6 trials, 1 hour apart in a light-colored tank filled with opaque water whitened by the addition of non-toxic paint at a temperature of 24.0±1.0°C. In the visible portion of the protocol, animals were trained to find a 12 × 12-cm submerged platform (1 cm below water surface) marked with a colored pole that served as a landmark placed in different quadrants of the pool. The animals were released at different locations in each 60-second trial. If mice did not find the platform in 60 seconds, they were gently guided to it. After remaining on the platform for 20 seconds, the animals were removed and placed in a dry cage under a warm heating lamp. Twenty m...
Mice were euthanized by isoflurane overdose followed by cervical dislocation. Hemibrains were flash frozen. One hemibrain was homogenized in liquid N 2 while the other was used in immunohistochemical determinations (5-6 per group) and for hippocampal dissections (5-6 per group). Half brains were microdissected to isolate the hippocampus by peeling away the cortex from the underlying hippocampus and releasing the hippocampus from the surrounding tissue, in particular the fimbria, by using fine surgical tweezers (Fine Science Tools) and then lifting toward the midline. The hippocampus separates easily but does include some adjacent white matter, which is then carefully tweezed off. We also obtained hippocampal tissue from at least 12 × 10 µm unfixed frozen sections mounted on glass slides. All but the hippocampal area was removed using a scalpel under a SZ60 Olympus dissecting microscope. The hippocampal tissue itself was removed by beading 10 µl of RIPA lysis buffer (25 mM Tris-HCl, pH 7.6; 150 mM NaCl; 1% NP40; 1% sodium deoxycholate; 0.1% sodium dodecyl sulfate) on it and then pipetting it up. For Western blot analyses, proteins from soluble fractions o...
Machine-readable layer
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