Neuronal activity regulates extracellular tau in vivo methods
Aim. Evidence-backed execution summary for Neuronal activity regulates extracellular tau in vivo methods from Neuronal activity regulates extracellular tau in vivo.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Animals.
reagent used in the protocol.
- Use
- All animal studies performed were reviewed and approved by the Animal Studies Committee at Washington University. 3-5-mo-old male and female P301S human tau transgenic mice and wild-type littermates on B6C3 background were used for microdialysis studies. Regulatable transgenic mice expressing anti-aggregant hu...
In vivo microdialysis.
reagent used in the protocol.
- Use
- In vivo microdialysis experiments to assess brain ISF tau levels from awake and freely moving mice were developed with modifications of our previously described method ( ). A guide cannula (Eicom microdialysis) was stereotactically implanted in the left hippocampus under isoflurane anesthesia, and cemented. After im...
Tau ELISA.
reagent used in the protocol.
- Use
- The tau concentration in media of brain slice culture and ISF of wild-type mice was analyzed in a tau sandwich ELISA with Tau-5 as a coating antibody (gift from L. Binder, Northwestern University, Evanston, IL) and biotinylated BT-2 (Thermo Fisher Scientific) as a detection antibody as previously described ( ). Huma...
Aβx-40 ELISA.
reagent used in the protocol.
- Use
- Aβx-40 ELISA was done as previously described ( ). Background determined by signal from the microdialysis perfusion buffer was subtracted.
Brain extraction.
reagent used in the protocol.
- Use
- Mice were perfused with chilled PBS-heparin. Brains were dissected for biochemical analysis and kept at -80°C until analyzed. To analyze tau after PTX infusion, left (exposed to PTX by reverse microdialysis) and right hippocampi were dissected at 4.5 h after PTX infusion where both ISF lactate and tau sho...
LDH assay.
reagent used in the protocol.
- Use
- LDH activity in ISF was determined by Lactate Dehydrogenase Activity Assay kit (Sigma-Aldrich). The final measurement [(A450)final] for calculating the enzyme activity was normalized by the baseline activities. After normalization, mean LDH activity during the 6-h PTX treatment and in the last 6-h pilocarpine treatm...
In vivo microdialysis.
In vivo microdialysis experiments to assess brain ISF tau levels from awake and freely moving mice were developed with modifications of our previously described method ( ). A guide cannula (Eicom microdialysis) was stereotactically implanted in the left hippocampus under isoflurane anesthesia, and cemented. After im...
- Use
- In vivo microdialysis experiments to assess brain ISF tau levels from awake and freely moving mice were developed with modifications of our previously described method ( ). A guide cannula (Eicom microdialysis) was stereotactically implanted in the left hippocampus under isoflurane anesthesia, and cemented. After im...
EEG recording.
Electrical activity was recorded in the hippocampus surrounding the microdialysis probe similar to. In brief, bipolar recording electrodes (Teflon-coated, stainless steel wire, 0.0055-in coated OD; A-M Systems) were attached to the shaft of the microdialysis guide cannula (BR-style; Bioanalytical) with Super-Fast E...
- Use
- Electrical activity was recorded in the hippocampus surrounding the microdialysis probe similar to. In brief, bipolar recording electrodes (Teflon-coated, stainless steel wire, 0.0055-in coated OD; A-M Systems) were attached to the shaft of the microdialysis guide cannula (BR-style; Bioanalytical) with Super-Fast E...
Glucose and lactate measurements.
Glucose and lactate concentration in ISF and media was determined by YSI2700 biochemistry analyzer (YSI Life Sciences). Glucose was used as a marker of substrate utilization, and lactate was used as a marker of neuronal activity ( ).
- Use
- Glucose and lactate concentration in ISF and media was determined by YSI2700 biochemistry analyzer (YSI Life Sciences). Glucose was used as a marker of substrate utilization, and lactate was used as a marker of neuronal activity ( ).
Statistical analysis.
Data in figures represent mean ± SEM. All statistical analysis was performed using Prism (version 5.04 for Windows; GraphPad Software). Analysis of the effects of drugs compared with vehicle control was done by two-way ANOVA with repeated measures. The comparison between baseline and post-treatment was done by...
- Use
- Data in figures represent mean ± SEM. All statistical analysis was performed using Prism (version 5.04 for Windows; GraphPad Software). Analysis of the effects of drugs compared with vehicle control was done by two-way ANOVA with repeated measures. The comparison between baseline and post-treatment was done by...
Statistical analysis.
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Data in figures represent mean ± SEM. All statistical analysis was performed using Prism (version 5.04 for Windows; GraphPad Software). Analysis of the effects of drugs compared with vehicle control was done by two-way ANOVA with repeated measures. The comparison between baseline and post-treatment was done by...
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The in vivo turnover rate of tau
To examine the clearance of soluble tau in vivo, the turnover rate of tau was kinetically analyzed using transgenic mice, which express a non-aggregating form of human tau (anti-aggregant mice: 2N4R-tau with ΔK280/PP mutations). The expression of human tau in these mice can be quickly switched off by doxycycline ( ). These mice do not develop tau pathology, allowing us to examine the clearance of soluble tau without the influence of tau aggregates (; ). To be able to analyze the elimination of ISF tau chronically for up to 18 d by microdialysis, mice were divided into five groups where human tau expression was switched off for the predetermined length of time. One cohort of mice was not exposed to doxycycline to determine the initial human tau levels before tau switch-off. In vivo microdialysis was performed in all groups and levels of ISF tau were assessed over 2 d in each mous...
In vivo microdialysis.
In vivo microdialysis experiments to assess brain ISF tau levels from awake and freely moving mice were developed with modifications of our previously described method ( ). A guide cannula (Eicom microdialysis) was stereotactically implanted in the left hippocampus under isoflurane anesthesia, and cemented. After implantation of the cannula and dummy probes (Eicom microdialysis), mice were habituated to microdialysis cages for one more day. After this recovery period, a 2-mm 1,000-kD cut-off AtmosLM microdialysis probe (Eicom) was inserted through the guide cannula. A probe was connected to a microdialysis peristaltic pump with two channels (MAB20; SciPro), which was operated in a push-pull mode. As a perfusion buffer, 25% human albumin solution (Gemini Bio Inc.) was diluted to 4% with artificial CSF (aCSF; 1.3 mM CaCl 2, 1.2 mM MgSO 4, 3 mM KCl, 0.4 mM KH 2 PO 4, 25 mM NaHCO 3, a...
EEG recording.
Electrical activity was recorded in the hippocampus surrounding the microdialysis probe similar to. In brief, bipolar recording electrodes (Teflon-coated, stainless steel wire, 0.0055-in coated OD; A-M Systems) were attached to the shaft of the microdialysis guide cannula (BR-style; Bioanalytical) with Super-Fast Epoxy Resin (Elmer's). Electrodes extended ∼1 mm from the tip of the guide, so that the electrode tip was located at the center of the 2-mm microdialysis membrane. EEG activity was assessed using a P511K A.C. pre-amplifier (Grass Instruments), digitized with a DigiData 1440 Data Acquisition System (Molecular Devices), and recorded digitally using Axoscope 10.2. Compounds were administered directly to the brain via reverse microdialysis at a flow rate of 1 µl/min.
Switch-off experiments.
16-17-mo-old transgenic mice expressing anti-aggregant human full-length tau (deltaK280/PP) were randomly divided into five groups. One cohort of mice was not exposed to doxycycline-containing food pellets (200 mg/kg; Bio-serv) and ISF was collected for 48 h (0 day groups). Other cohorts of mice received doxycycline-containing food pellets for 2, 6, 8, or 16 d, respectively before microdialysis. On day 2, 6, 8, or 16, in vivo microdialysis was started and ISF samples were collected for 48 h. During microdialysis, mice were given doxycycline-containing food pellets. Brains were collected on day 4, 8, 10, or 18, respectively, at the end of microdialysis experiments. Hippocampus where microdialysis probes were inserted was used to determine intracellular human tau levels in hippocampus. Percentages of brain human tau, ISF human tau, and ISF lactate were normalized by the mean conce...
Pilocarpine injection.
Mice received a low dose of methyl scopolamine (1 mg/kg) to reduce peripheral cholinergic effects. After 30 min, mice were given i.p. injection of 325 mg/kg pilocarpine hydrochloride. All animals injected with pilocarpine displayed motor seizure phenotype. 15 min after pilocarpine injection, the mice were injected with pentobarbital to terminate seizures.
Brain extraction.
Mice were perfused with chilled PBS-heparin. Brains were dissected for biochemical analysis and kept at -80°C until analyzed. To analyze tau after PTX infusion, left (exposed to PTX by reverse microdialysis) and right hippocampi were dissected at 4.5 h after PTX infusion where both ISF lactate and tau showed the highest increases from baseline. Tau in hippocampal lysates was determined by Tau-5/BT2 ELISA. Contralateral hippocampus (right) was used as a control. In switch-off experiments, hippocampus was weighed and homogenized in RAB buffer (100 mM MES, 1 mM EDTA, 0.5 mM MgSO 4, 750 mM NaCl, 20 mM NaF, and 1 mM Na 3 VO 4, supplemented by protease inhibitors [Complete; Roche] and phosphatase inhibitors [PhosSTOP; Roche]). The samples were centrifuged at 50,000 g for 20 min at 4°C. The supernatants were collected as RAB soluble fractions. The pellets were further homog...
Measurement outputs
What raw and processed outputs should exist?
To examine the clearance of soluble tau in vivo, the turnover rate of tau was kinetically analyzed using transgenic mice, which express a non-aggregating form of human tau (anti...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The turnover rate of tau in hippocampus and ISF is low. Human tau expression in anti-aggregant mice was switched off for the indicated lengths of time by doxycycline, and human...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
All animal studies performed were reviewed and approved by the Animal Studies Committee at Washington University. 3-5-mo-old male and female P301S human tau transgenic mic...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
In vivo microdialysis experiments to assess brain ISF tau levels from awake and freely moving mice were developed with modifications of our previously described method ( ). A gu...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
TTX does not cause an acute decrease of ISF tau.
from paperScoring or quantification
Quantify the primary readouts for this experiment: To examine the clearance of soluble tau in vivo, the turnover rate of tau was kinetically analyzed using transgenic mice, which express a non-aggregating form of human tau (anti...; The turnover rate of tau in hippocampus and ISF is low. Human tau expression in anti-aggregant mice was switched off for the indicated lengths of time by doxycycline, and human...; All animal studies performed were reviewed and approved by the Animal Studies Committee at Washington University. 3-5-mo-old male and female P301S human tau transgenic mic...; In vivo microdialysis experiments to assess brain ISF tau levels from awake and freely moving mice were developed with modifications of our previously described method ( ). A gu....
from paperStatistical comparison
TTX does not cause an acute decrease of ISF tau. After baseline ISF tau collection, 5 µM TTX was delivered via reverse microdialysis (indicated by dashed line). The effect...; The turnover rate of tau in hippocampus and ISF is low. Human tau expression in anti-aggregant mice was switched off for the indicated lengths of time by doxycycline, and human...; Data in figures represent mean ± SEM. All statistical analysis was performed using Prism (version 5.04 for Windows; GraphPad Software). Analysis of the effects of drugs com...
from paperReporting output
Report representative outputs alongside summary comparisons for To examine the clearance of soluble tau in vivo, the turnover rate of tau was kinetically analyzed using transgenic mice, which express a non-aggregating form of human tau (anti..., The turnover rate of tau in hippocampus and ISF is low. Human tau expression in anti-aggregant mice was switched off for the indicated lengths of time by doxycycline, and human..., All animal studies performed were reviewed and approved by the Animal Studies Committee at Washington University. 3-5-mo-old male and female P301S human tau transgenic mic..., In vivo microdialysis experiments to assess brain ISF tau levels from awake and freely moving mice were developed with modifications of our previously described method ( ). A gu....
inferred from protocolStructured statistical methods
TTX does not cause an acute decrease of ISF tau. After baseline ISF tau collection, 5 µM TTX was delivered via reverse microdialysis (indicated by dashed line). The effect...; The turnover rate of tau in hippocampus and ISF is low. Human tau expression in anti-aggregant mice was switched off for the indicated lengths of time by doxycycline, and human...; Data in figures represent mean ± SEM. All statistical analysis was performed using Prism (version 5.04 for Windows; GraphPad Software). Analysis of the effects of drugs com...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (6)
To examine the clearance of soluble tau in vivo, the turnover rate of tau was kinetically analyzed using transgenic mice, which express a non-aggregating form of human tau (anti-aggregant mice: 2N4R-tau with ΔK280/PP mutations). The expression of human tau in these mice can be quickly switched off by doxycycline ( ). These mice do not develop tau pathology, allowing us to examine the clearance of soluble tau without the influence of tau aggregates (; ). To be able to analyze the elimination of ISF tau chronically for up to 18 d by microdialysis, mice were divided into five groups where human tau expression was switched off for the predetermined length of time. One cohort of mice was not exposed to doxycycline to determine the initial human tau levels before tau switch-off. In vivo microdialysis was performed in all groups and levels of ISF tau were assessed over 2 d in each mouse. ISF human tau levels were determined by ELISA. The absolute ISF human tau levels after switch-off were normalized to the level of ISF human tau in the mice without switch-off. Soluble human tau in the hippocampus where ISF tau was collected was also analyzed at the end of microdialysis.
In vivo microdialysis experiments to assess brain ISF tau levels from awake and freely moving mice were developed with modifications of our previously described method ( ). A guide cannula (Eicom microdialysis) was stereotactically implanted in the left hippocampus under isoflurane anesthesia, and cemented. After implantation of the cannula and dummy probes (Eicom microdialysis), mice were habituated to microdialysis cages for one more day. After this recovery period, a 2-mm 1,000-kD cut-off AtmosLM microdialysis probe (Eicom) was inserted through the guide cannula. A probe was connected to a microdialysis peristaltic pump with two channels (MAB20; SciPro), which was operated in a push-pull mode. As a perfusion buffer, 25% human albumin solution (Gemini Bio Inc.) was diluted to 4% with artificial CSF (aCSF; 1.3 mM CaCl 2, 1.2 mM MgSO 4, 3 mM KCl, 0.4 mM KH 2 PO 4, 25 mM NaHCO 3, and 122 mM NaCl, pH 7.35) on the day of use and filtered through a 0.1-µm membrane. For 100 mM high K + stimulation, 97 mM KCl in aCSF was substituted for an equal amount of NaCl. Normal perfusion buffer was switched to high K + perfusion buffer for 1 h. Before microdialysis sample collection, a...
Electrical activity was recorded in the hippocampus surrounding the microdialysis probe similar to. In brief, bipolar recording electrodes (Teflon-coated, stainless steel wire, 0.0055-in coated OD; A-M Systems) were attached to the shaft of the microdialysis guide cannula (BR-style; Bioanalytical) with Super-Fast Epoxy Resin (Elmer's). Electrodes extended ∼1 mm from the tip of the guide, so that the electrode tip was located at the center of the 2-mm microdialysis membrane. EEG activity was assessed using a P511K A.C. pre-amplifier (Grass Instruments), digitized with a DigiData 1440 Data Acquisition System (Molecular Devices), and recorded digitally using Axoscope 10.2. Compounds were administered directly to the brain via reverse microdialysis at a flow rate of 1 µl/min.
16-17-mo-old transgenic mice expressing anti-aggregant human full-length tau (deltaK280/PP) were randomly divided into five groups. One cohort of mice was not exposed to doxycycline-containing food pellets (200 mg/kg; Bio-serv) and ISF was collected for 48 h (0 day groups). Other cohorts of mice received doxycycline-containing food pellets for 2, 6, 8, or 16 d, respectively before microdialysis. On day 2, 6, 8, or 16, in vivo microdialysis was started and ISF samples were collected for 48 h. During microdialysis, mice were given doxycycline-containing food pellets. Brains were collected on day 4, 8, 10, or 18, respectively, at the end of microdialysis experiments. Hippocampus where microdialysis probes were inserted was used to determine intracellular human tau levels in hippocampus. Percentages of brain human tau, ISF human tau, and ISF lactate were normalized by the mean concentration in 0 day groups.
Mice received a low dose of methyl scopolamine (1 mg/kg) to reduce peripheral cholinergic effects. After 30 min, mice were given i.p. injection of 325 mg/kg pilocarpine hydrochloride. All animals injected with pilocarpine displayed motor seizure phenotype. 15 min after pilocarpine injection, the mice were injected with pentobarbital to terminate seizures.
Mice were perfused with chilled PBS-heparin. Brains were dissected for biochemical analysis and kept at -80°C until analyzed. To analyze tau after PTX infusion, left (exposed to PTX by reverse microdialysis) and right hippocampi were dissected at 4.5 h after PTX infusion where both ISF lactate and tau showed the highest increases from baseline. Tau in hippocampal lysates was determined by Tau-5/BT2 ELISA. Contralateral hippocampus (right) was used as a control. In switch-off experiments, hippocampus was weighed and homogenized in RAB buffer (100 mM MES, 1 mM EDTA, 0.5 mM MgSO 4, 750 mM NaCl, 20 mM NaF, and 1 mM Na 3 VO 4, supplemented by protease inhibitors [Complete; Roche] and phosphatase inhibitors [PhosSTOP; Roche]). The samples were centrifuged at 50,000 g for 20 min at 4°C. The supernatants were collected as RAB soluble fractions. The pellets were further homogenized by RIPA buffer (150 mm NaCl, 50 mm Tris, 0.5% deoxycholic acid, 1% Triton X-100, and 0.5% SDS-25 mm EDTA, pH 8.0, supplemented by protease inhibitor [Complete; Roche] and phosphatase inhibitor [PhosSTOP; Roche]). Total tau in both RAB and RIPA soluble fractions were combined and used as...
Machine-readable layer
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"text": "Mice were perfused with chilled PBS-heparin. Brains were dissected for biochemical analysis and kept at -80°C until analyzed. To analyze tau after PTX infusion, left (exposed to PTX by reverse microdialysis) and right hippocampi were dissected at 4.5 h after PTX infusion where both ISF lactate and tau showed the highest increases from baseline. Tau in hippocampal lysates was determined by Tau-5/BT2 ELISA. Contralateral hippocampus (right) was used as a control. In switch-off experiments, hippocampus was weighed and homogenized in RAB buffer (100 mM MES, 1 mM EDTA, 0.5 mM MgSO 4, 750 mM NaCl, 20 mM NaF, and 1 mM Na 3 VO 4, supplemented by protease inhibitors [Complete; Roche] and phosphatase inhibitors [PhosSTOP; Roche]). The samples were centrifuged at 50,000 g for 20 min at 4°C. The supernatants were collected as RAB soluble fractions. The pellets were further homog..."
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