Neuronal activity regulates extracellular tau in vivo methods
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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...
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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...
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