Environmental enrichment strengthens corticocortical interactions and reduces amyloid-β oligomers in aged mice methods
Aim. Evidence-backed execution summary for Environmental enrichment strengthens corticocortical interactions and reduces amyloid-β oligomers in aged mice methods from Environmental enrichment strengthens corticocortical interactions and reduces amyloid-β oligomers in aged mice.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Neuronatomical tracing
reagent used in the protocol.
- Use
- Monosynaptic connections between the primary visual (V1) and auditory cortices (A1) were identified using the retrograde tracer Cholera Toxin β subunit (CTB, Sigma, USA). Mice were mounted on a stereotaxic apparatus, then a burr hole was made in the skull overlying V1. Injections were performed in the core of V...
Chronic electrode implants and LFP recordings
reagent used in the protocol.
- Use
- After a 1 h habituation to the test cage, LFPs were recorded for 1 h using a digital acquisition system, composed of a custom-made buffer to eliminate movement artifacts, an amplifier and an acquisition card (National Instruments, USA), plugged via USB to a computer. The custom-made acquisition software was based on...
Western blotting
reagent used in the protocol.
- Use
- After chloral hydrate anaesthesia, the brain was removed from the skull and the entire cortical mantle was dissected out, frozen in dry ice and stored at -80°C. Samples were homogenated and soluble proteins were extracted with a lysis buffer adapted from Lesne et al. ( ), that contained 0.01% NP-40, 0.1%...
Environmental enrichment modulates levels of soluble amyloid beta oligomers
reagent used in the protocol.
- Use
- Since Aβ aggregates are observed across a wide range of molecular weights, with specific immunoreactivity profiles to antibodies commonly used in western blotting (Lesne et al., ), we performed Western blot analyses using two different antibodies, namely A11 and 4G8 (Table ). These antibodies were selected beca...
Environmental enrichment modulates levels of soluble amyloid beta oligomers
reagent used in the protocol.
- Use
- Decrease of amyloid β oligomers in aged enriched mice. (A) Left, representative immunoblotting and right, quantification of the expression of Aβ oligomers probed with the A11 polyclonal antibody. (B) Left, representative immunoblotting and right, quantification of expression of Aβ oligomers probed wit...
Materials and methods
C57BL/6J male mice were housed in an animal room with a 12h/12h light/dark cycle, with food and water available ad libitum. The standard rearing condition (SC) consisted of a 26 × 18 × 18 cm cage housing 3 animals. The environmental enrichment condition (EE) consisted of a large cage (44 × 62 ×...
- Use
- C57BL/6J male mice were housed in an animal room with a 12h/12h light/dark cycle, with food and water available ad libitum. The standard rearing condition (SC) consisted of a 26 × 18 × 18 cm cage housing 3 animals. The environmental enrichment condition (EE) consisted of a large cage (44 × 62 ×...
Neuronatomical tracing
Monosynaptic connections between the primary visual (V1) and auditory cortices (A1) were identified using the retrograde tracer Cholera Toxin β subunit (CTB, Sigma, USA). Mice were mounted on a stereotaxic apparatus, then a burr hole was made in the skull overlying V1. Injections were performed in the core of V...
- Use
- Monosynaptic connections between the primary visual (V1) and auditory cortices (A1) were identified using the retrograde tracer Cholera Toxin β subunit (CTB, Sigma, USA). Mice were mounted on a stereotaxic apparatus, then a burr hole was made in the skull overlying V1. Injections were performed in the core of V...
Chronic electrode implants and LFP recordings
Local field potential (LFP) recordings were performed in awake, freely moving mice using an adaptation of the protocol described by Mainardi et al. ( ).
- Use
- Local field potential (LFP) recordings were performed in awake, freely moving mice using an adaptation of the protocol described by Mainardi et al. ( ).
Chronic electrode implants and LFP recordings
Under avertin anaesthesia (0.01 ml/g) and after placement in a stereotaxic apparatus, the skull was exposed and four burr holes were drilled (see Figure and below), paying attention not to damage the dural surface. Four 120 µm-thick nichrome wire electrodes and an insulated copper ground cable were soldered to...
- Use
- Under avertin anaesthesia (0.01 ml/g) and after placement in a stereotaxic apparatus, the skull was exposed and four burr holes were drilled (see Figure and below), paying attention not to damage the dural surface. Four 120 µm-thick nichrome wire electrodes and an insulated copper ground cable were soldered to...
Chronic electrode implants and LFP recordings
After a 1 h habituation to the test cage, LFPs were recorded for 1 h using a digital acquisition system, composed of a custom-made buffer to eliminate movement artifacts, an amplifier and an acquisition card (National Instruments, USA), plugged via USB to a computer. The custom-made acquisition software was based on...
- Use
- After a 1 h habituation to the test cage, LFPs were recorded for 1 h using a digital acquisition system, composed of a custom-made buffer to eliminate movement artifacts, an amplifier and an acquisition card (National Instruments, USA), plugged via USB to a computer. The custom-made acquisition software was based on...
Western blotting
After chloral hydrate anaesthesia, the brain was removed from the skull and the entire cortical mantle was dissected out, frozen in dry ice and stored at -80°C. Samples were homogenated and soluble proteins were extracted with a lysis buffer adapted from Lesne et al. ( ), that contained 0.01% NP-40, 0.1%...
- Use
- After chloral hydrate anaesthesia, the brain was removed from the skull and the entire cortical mantle was dissected out, frozen in dry ice and stored at -80°C. Samples were homogenated and soluble proteins were extracted with a lysis buffer adapted from Lesne et al. ( ), that contained 0.01% NP-40, 0.1%...
Statistical analyses
All the analyses have been performed using the SigmaPlot 12 software (SyStat Software, USA).
- Use
- All the analyses have been performed using the SigmaPlot 12 software (SyStat Software, USA).
EE modulates the spectral power of local field potentials in V1 and A1
After identifying a monosynaptic connection between A1 and V1, we asked whether EE could modulate (i) the profile of local activities in these two primary sensory areas and (ii) the interactions between them, by performing multichannel recordings of local field potentials (LFPs) from chronically implanted electrodes...
- Use
- After identifying a monosynaptic connection between A1 and V1, we asked whether EE could modulate (i) the profile of local activities in these two primary sensory areas and (ii) the interactions between them, by performing multichannel recordings of local field potentials (LFPs) from chronically implanted electrodes...
Statistical analyses
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All the analyses have been performed using the SigmaPlot 12 software (SyStat Software, USA).
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Materials and methods
C57BL/6J male mice were housed in an animal room with a 12h/12h light/dark cycle, with food and water available ad libitum. The standard rearing condition (SC) consisted of a 26 × 18 × 18 cm cage housing 3 animals. The environmental enrichment condition (EE) consisted of a large cage (44 × 62 × 28 cm) containing one running wheel for voluntary physical exercise and differently shaped objects (tunnels, shelters, stairs) that were repositioned twice a week and completely substituted once a week. Six to eight mice were housed in EE cages. Aged mice (age 17 months, EE-OLD group) were placed in EE for one month after electrode implantation, then brain samples were collected (see below). Age-matched mice reared in SC were used as controls (SC-OLD group). For the experiments on young mice (EE-YOUNG and SC-YOUNG groups), pregnant dams were put either in EE 1 week before d...
Neuronatomical tracing
Monosynaptic connections between the primary visual (V1) and auditory cortices (A1) were identified using the retrograde tracer Cholera Toxin β subunit (CTB, Sigma, USA). Mice were mounted on a stereotaxic apparatus, then a burr hole was made in the skull overlying V1. Injections were performed in the core of V1, 0.0 mm anteroposterior (AP) and 2.5 mm lateral (L) to the lambda point (Paxinos and Franklin, ). Only 50 nl of CTB solution (1% in water) at 600 µm depth were delivered using a 1 µl Hamilton syringe (Hamilton, USA) and a glass pipette, thus avoiding spilling of CTB outside of V1. After allowing 3 days for CTB transport to neuronal somata and processes, animals received terminal anaesthesia with chloral hydrate and transcardial perfusion with 25 ml of PBS followed by 50 ml of 4% PFA in 0.1 M phosphate buffer pH 7.4 (PB). Brains were quickly removed, cryoprotecte...
Chronic electrode implants and LFP recordings
Under avertin anaesthesia (0.01 ml/g) and after placement in a stereotaxic apparatus, the skull was exposed and four burr holes were drilled (see Figure and below), paying attention not to damage the dural surface. Four 120 µm-thick nichrome wire electrodes and an insulated copper ground cable were soldered to a multipin socket. This device was held by an adjustable manipulator and the electrodes were positioned to obtain an electrical contact without lesioning the dura mater. LFPs were sampled by placing in each cortical area two electrodes, spaced by 1.0 mm, to detect local electrical activity between the two sites. A screw was positioned on the occipital bone and connected with the ground cable, while an additional screw was installed on the frontal bone for improved stability. The implant was fixed with acrylic cement (Paladur, Pala, Germany). Stereotaxical coordinates were (...
Chronic electrode implants and LFP recordings
After a 1 h habituation to the test cage, LFPs were recorded for 1 h using a digital acquisition system, composed of a custom-made buffer to eliminate movement artifacts, an amplifier and an acquisition card (National Instruments, USA), plugged via USB to a computer. The custom-made acquisition software was based on LabView (National Instruments). Cortical LFPs were sampled at 100 Hz as the differential between two adjacent electrode sites, 10000 × amplified and 0.3-45 Hz band-passed.
LFP analysis
Data sets consisted of bivariate time series corresponding to LFPs simultaneously recorded from V1 and A1. Frequency band decomposition of LFP signals was performed considering the main EEG frequency bands: δ (0.3-4 Hz), θ (4-8 Hz), α (8-12 Hz), β (12-30 Hz) and low-γ (30-45 Hz), which were isolated by band-pass filtering the original LFP signal using a custom-made application based on LabView. Each time series was normalized to zero mean and unit standard deviation. To satisfy the request of stationarity all-time series were partitioned in half-overlapping windows.
Cross correlation
The correlation level of LFP signals from V1 and A1 was quantified using a measure based on the cross-correlation function (CC) (Mormann et al., ), with an adaptation of the protocol we described in Di Garbo et al. ( ).
Cross correlation
To quantify the interdependence properties between the LFPs in V1 and A1, we employed the following measure: ρ m = max j | ρ ( j ) | where j = 1, 2,.., N Lag is the number of time lags. ρ m quantifies the higher value of the linear correlation between two signals over the time windows N Lag Δ t s. The adopted value for N Lag was 3, which represents a compromise between computational advantage and the choice of a physiological time window. For each experimental group the set of all values of ρ m was obtained; then, the corresponding mean ± s.e.m. values were calculated.
Cross correlation
The CC analysis described above was employed both on the original LFP signals (0.3-45 Hz) and on the δ, θ, α, β, and low-γ bands (see above, Data Description).
Measurement outputs
What raw and processed outputs should exist?
Data sets consisted of bivariate time series corresponding to LFPs simultaneously recorded from V1 and A1. Frequency band decomposition of LFP signals was performed considering...
- 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
Spectral analysis of LFPs was performed using the Fast Fourier Transform (FFT) algorithm (Bendat and Persol, ). LFP series were divided in half-overlapping windows of 8192 data...
- 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
The CC analysis described above was employed both on the original LFP signals (0.3-45 Hz) and on the δ, θ, α, β, and low-γ bands (see above, Data...
- 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
Antibodies, dilutions and amount of protein loaded in Western blot assays.
- 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
For analysis of LFP signals, a total of 35 mice were used, 13 for the EE-OLD group, 10 for the SC-OLD group, 6 for the EE-YOUNG group and 6 for the SC-YOUNG group.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Data sets consisted of bivariate time series corresponding to LFPs simultaneously recorded from V1 and A1. Frequency band decomposition of LFP signals was performed considering...; Spectral analysis of LFPs was performed using the Fast Fourier Transform (FFT) algorithm (Bendat and Persol, ). LFP series were divided in half-overlapping windows of 8192 data...; The CC analysis described above was employed both on the original LFP signals (0.3-45 Hz) and on the δ, θ, α, β, and low-γ bands (see above, Data...; Antibodies, dilutions and amount of protein loaded in Western blot assays..
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
For analysis of LFP signals, a total of 35 mice were used, 13 for the EE-OLD group, 10 for the SC-OLD group, 6 for the EE-YOUNG group and 6 for the SC-YOUNG group. For comparing...; For Western blots on vGluT-1 and vGAT (Figure ), a total of 26 mice were used, 12 for the EE-OLD group, 9 for SC-OLD group and 5 for the SC-YOUNG group. Statistical significance...; For Western blots on amyoid-β oligomers and neprilysin (Figures, ), a total of 36 mice were used, 22 for the EE-OLD group and 14 for the SC-OLD group. Statistical signific...; All the analyses have been performed using the SigmaPlot 12 software (SyStat Software, USA).
from paperReporting output
Report representative outputs alongside summary comparisons for Data sets consisted of bivariate time series corresponding to LFPs simultaneously recorded from V1 and A1. Frequency band decomposition of LFP signals was performed considering..., Spectral analysis of LFPs was performed using the Fast Fourier Transform (FFT) algorithm (Bendat and Persol, ). LFP series were divided in half-overlapping windows of 8192 data..., The CC analysis described above was employed both on the original LFP signals (0.3-45 Hz) and on the δ, θ, α, β, and low-γ bands (see above, Data..., Antibodies, dilutions and amount of protein loaded in Western blot assays..
inferred from protocolStructured statistical methods
For analysis of LFP signals, a total of 35 mice were used, 13 for the EE-OLD group, 10 for the SC-OLD group, 6 for the EE-YOUNG group and 6 for the SC-YOUNG group. For comparing...; For Western blots on vGluT-1 and vGAT (Figure ), a total of 26 mice were used, 12 for the EE-OLD group, 9 for SC-OLD group and 5 for the SC-YOUNG group. Statistical significance...; For Western blots on amyoid-β oligomers and neprilysin (Figures, ), a total of 36 mice were used, 22 for the EE-OLD group and 14 for the SC-OLD group. Statistical signific...; All the analyses have been performed using the SigmaPlot 12 software (SyStat Software, USA).
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
C57BL/6J male mice were housed in an animal room with a 12h/12h light/dark cycle, with food and water available ad libitum. The standard rearing condition (SC) consisted of a 26 × 18 × 18 cm cage housing 3 animals. The environmental enrichment condition (EE) consisted of a large cage (44 × 62 × 28 cm) containing one running wheel for voluntary physical exercise and differently shaped objects (tunnels, shelters, stairs) that were repositioned twice a week and completely substituted once a week. Six to eight mice were housed in EE cages. Aged mice (age 17 months, EE-OLD group) were placed in EE for one month after electrode implantation, then brain samples were collected (see below). Age-matched mice reared in SC were used as controls (SC-OLD group). For the experiments on young mice (EE-YOUNG and SC-YOUNG groups), pregnant dams were put either in EE 1 week before delivery together with 2-3 non-pregnant helper females, or left in SC, in the latter case with no helper females. Pups were weaned at postnatal day (P) 25.
Monosynaptic connections between the primary visual (V1) and auditory cortices (A1) were identified using the retrograde tracer Cholera Toxin β subunit (CTB, Sigma, USA). Mice were mounted on a stereotaxic apparatus, then a burr hole was made in the skull overlying V1. Injections were performed in the core of V1, 0.0 mm anteroposterior (AP) and 2.5 mm lateral (L) to the lambda point (Paxinos and Franklin, ). Only 50 nl of CTB solution (1% in water) at 600 µm depth were delivered using a 1 µl Hamilton syringe (Hamilton, USA) and a glass pipette, thus avoiding spilling of CTB outside of V1. After allowing 3 days for CTB transport to neuronal somata and processes, animals received terminal anaesthesia with chloral hydrate and transcardial perfusion with 25 ml of PBS followed by 50 ml of 4% PFA in 0.1 M phosphate buffer pH 7.4 (PB). Brains were quickly removed, cryoprotected in 30% W/V sucrose in PB for 3 days, then 50 µm-thick coronal sections were obtained with a sliding microtome (Leica, Germany). CTB labeling was visualized by immunohistochemistry. Free-floating sections were blocked in 5% normal rabbit serum (NRS), 2.5% bovine serum albumin (BSA), 0.3% Trito...
Under avertin anaesthesia (0.01 ml/g) and after placement in a stereotaxic apparatus, the skull was exposed and four burr holes were drilled (see Figure and below), paying attention not to damage the dural surface. Four 120 µm-thick nichrome wire electrodes and an insulated copper ground cable were soldered to a multipin socket. This device was held by an adjustable manipulator and the electrodes were positioned to obtain an electrical contact without lesioning the dura mater. LFPs were sampled by placing in each cortical area two electrodes, spaced by 1.0 mm, to detect local electrical activity between the two sites. A screw was positioned on the occipital bone and connected with the ground cable, while an additional screw was installed on the frontal bone for improved stability. The implant was fixed with acrylic cement (Paladur, Pala, Germany). Stereotaxical coordinates were (i) 2.0 mm and 3.0 mm L and 0.0 mm AP to lambda for V1; (ii) 3.9 mm L and -2.0 and -3.0 mm AP to bregma for primary auditory cortex (A1) (Figure ) (Paxinos and Franklin, ). Five days were allowed for recovery from surgery.
After a 1 h habituation to the test cage, LFPs were recorded for 1 h using a digital acquisition system, composed of a custom-made buffer to eliminate movement artifacts, an amplifier and an acquisition card (National Instruments, USA), plugged via USB to a computer. The custom-made acquisition software was based on LabView (National Instruments). Cortical LFPs were sampled at 100 Hz as the differential between two adjacent electrode sites, 10000 × amplified and 0.3-45 Hz band-passed.
Data sets consisted of bivariate time series corresponding to LFPs simultaneously recorded from V1 and A1. Frequency band decomposition of LFP signals was performed considering the main EEG frequency bands: δ (0.3-4 Hz), θ (4-8 Hz), α (8-12 Hz), β (12-30 Hz) and low-γ (30-45 Hz), which were isolated by band-pass filtering the original LFP signal using a custom-made application based on LabView. Each time series was normalized to zero mean and unit standard deviation. To satisfy the request of stationarity all-time series were partitioned in half-overlapping windows.
The correlation level of LFP signals from V1 and A1 was quantified using a measure based on the cross-correlation function (CC) (Mormann et al., ), with an adaptation of the protocol we described in Di Garbo et al. ( ).
To quantify the interdependence properties between the LFPs in V1 and A1, we employed the following measure: ρ m = max j | ρ ( j ) | where j = 1, 2,.., N Lag is the number of time lags. ρ m quantifies the higher value of the linear correlation between two signals over the time windows N Lag Δ t s. The adopted value for N Lag was 3, which represents a compromise between computational advantage and the choice of a physiological time window. For each experimental group the set of all values of ρ m was obtained; then, the corresponding mean ± s.e.m. values were calculated.
The CC analysis described above was employed both on the original LFP signals (0.3-45 Hz) and on the δ, θ, α, β, and low-γ bands (see above, Data Description).
Machine-readable layer
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