Double-mutant mice were produced carrying various combinations of Kcna1
- and Tau
- mice carry a null mutation in the Kcna1
gene on chromosome 6 (Smart et al., 1998
- mice contain a targeted knockout mutation in the Mapt
gene on chromosome 11 (Dawson et al., 2001
). Heterozygous F1 mice (Kcna1
+/−), derived by crossing heterozygous Kcna1
−/+ mice (Tac:N:NIHS-BC) with homozygous Tau
−/−(C57BL/6) mice, were intercrossed to produce F2 double mutants of mixed Black Swiss (BlSw, Tac:N:NIHS-BC) and C57BL/6 background. Mice of either sex were used for all experiments unless otherwise stated. Mice were housed at 22°C with a 12 h light/dark cycle and fed ad libitum. All procedures were carried out in accordance with the guidelines of the National Institute of Health, as approved by the Animal Care and Use Committee of Baylor College of Medicine.
Genomic DNA was isolated from tail clips using DirectPCR Lysis Reagent (Viagen Biotech). Genotype was determined using PCR amplification for specific alleles as described previously for Kcna1
- (Smart et al., 1998
) and Tau
- (Dawson et al., 2001
). PCR products were separated on a 1.5% agarose gel by electrophoresis.
In vivo video-electroencephalography (EEG) recordings
Kcna1−/− mice bred with varying tau alleles (Tau+/+, Tau+/−, and Tau−/−) were anesthetized by Avertin and surgically implanted with bilateral silver wire electrodes (0.005 inch diameter) attached to a microminiature connector. EEG wires were inserted into the subdural space over the temporal cortex through cranial burr holes. Mice were allowed to recover for 24 or more hours before analysis. EEG and behavioral activity in freely moving mice were analyzed using simultaneous video-EEG monitoring (Haramonie software version 6.1c, Stellate Systems). All EEG signals were filtered using a 0.3 Hz high-pass filter, 70 Hz low-pass filter, and 60 Hz notch filter. Eight mice of each genotype were monitored at 4–6 weeks of age and were each evaluated for a total of 9 or more hours. Mice were recorded in one or more 3–9 hour monitoring sessions over several days to mitigate the effect of seizure clustering. Seizure activity defined by EEG waveform and corresponding video-recorded behavior was quantified by visual inspection.
Hippocampal slice preparation and electrophysiology
Transverse hippocampal slices (300 μm thickness) from double-mutant mice of genotypes Kcna1
+/+, and Kcna1
−/− (6–11 weeks old) were prepared using a vibrotome. Slices were sectioned in cutting solution containing (in mM) 100 sucrose, 30 NaCl, 3 KCl, 0.5 CaCl2
, 28 NaHCO3
, 7 MgCl2
, 1.4 NaH2
, and 11 D-glucose and were constantly gassed with 95%O2
to maintain a constant pH of 7.4. After incubation in artificial CSF (ACSF) at 32°C for 1 h, slices were transferred into a submerged recording chamber for electrophysiological recordings. Brain slices were constantly perfused with ACSF containing (in mM) 125 NaCl, 2.5 KCl, 2 CaCl2
, 25 NaHCO3
, 1 MgCl, 1.25 NaH2
, and 11 D-glucose. Recording chamber temperature was controlled at 32–33°C. Recording pipettes (4–6 Mohm) were pulled from borosilicate glass and filled with 2M NaCl. CA3 pyramidal neurons have been previously shown to exhibit altered in vitro
network excitability in Kcna1
−/− mice (Smart et al., 1998
, Lopantsev et al., 2003
, Glasscock et al., 2007
). Therefore, field recordings were made from the CA3 pyramidal somata identified visually in the stratum pyramidale using a Getting Instruments Model 5A amplifier, digitized by a Digidata 1322A and collected using Clampex (Molecular Devices). A low pass filter was set at 5 kHz. Slices were perfused with ACSF containing 7.5 mM KCl (Glasscock et al., 2007
) and began synchronously discharging within a period of 15–25 minutes. To calculate the burst frequency, slices were allowed to equilibrate to 7.5 mM KCl for an additional 5–15 minutes and the burst frequency was then calculated over a 5 minute period. The burst duration was defined as the interval between baseline crossings and analyzed as the average of 10 bursts per slice. Data analysis was performed using Clampfit (Molecular Devices) and Origin 7.5 (OriginLab).
3-Dimensional Magnetic Resonance Imaging (3D-MRI) and brain volumetry
MRI of the brain was performed on 12-week-old male mice of genotypes Kcna1−/−Tau+/+, Kcna1−/−Tau−/−, Kcna1+/+Tau+/+, and Kcna1+/+Tau−/− (n=3). Mice were overdosed with isoflourane, placed in a 50mL conical tube, and all imaged identically within 1 h. To mitigate post mortem delay, mice were cooled to 20°C during imaging. MRI images were obtained using a 9.4T, 21 cm bore horizontal scanner with a 35 mm volume resonator (Bruker, BioSpin). The imaging parameters used to obtain three dimensional (3D) rapid acquisition with relaxation enhancement (RARE) images of the mouse brain were as follows: TR = 2000 ms; TE = 11.713 ms; Effective TE = 46.85 ms; FOV = 25 mm3; matrix = 256 × 256 × 164; RARE Factor = 8; Number of Averages = 5; Total Scan Time = 14 h 34 m 40 s. Images were obtained using Paravision software version 4 (Bruker, BioSpin).
MRI images were analyzed while blinded to genotype using Amira 3.1 software (Visage Imaging). The hippocampal border was segmented manually in both coronal and sagittal planes and the hippocampus volume measurement for each mouse was computed as the average of the volumes in the two planes. The forebrain volume was segmented in the sagittal plane.
Fly stocks and behavioral testing
were maintained on standard media at 25–26°C. The bang-sensitive (BS) strains used were easily shocked
which encodes an ethanolamine kinase, and kazachoc
which encodes a K+
cotransporter, and were obtained from M. Tanouye at University of California, Berkley (Pavlidis et al., 1994
, Hekmat-Scafe et al., 2006
). The BS alleles used in this study were easPC80
. Tau P-element (tauEP3203
) and deficiency (Df(3R) MR22
) alleles were obtained from Bloomington Stock Center and D. St. Johnston at University of Cambridge, respectively (Doerflinger et al., 2003
). Male and female kcc
flies and male eas
flies were analyzed for bang sensitivity 1–2 days post eclosion and not exposed to CO2
within 2 hours preceding testing. To test, no more than 10 flies were placed in a clean vial and allowed to rest for 30 minutes. Flies were vortexed (VWR) at maximum strength for 10 seconds and visually monitored for the presence of paralysis and seizure in each fly.
Kcna1−/− and Kcna1+/+ forebrain hemisphere samples (4.5 weeks old) were extracted and flash frozen in liquid nitrogen. Samples were homogenized on ice using a Tissue Tearor (VWR) in lysis buffer with phosphatase inhibitors (20 mM Tris, pH 7.5, 138 mM NaCl, 3 mM KCl, 1% Tx-100, 1 mM EGTA, 2 mM EDTA, 1 mM Benzamidine, 5 μg/ml Aprotinin, 5 μg/ml Leupeptin, 5 μg/ml Pepstatin A, 1 mM PMSF, 1 mM DTT, 50 mM NaF, 1 mM Na3VO4) and protein concentration determined by Pierce BCA Protein Assay Kit (Thermo Scientific). 20 μg of protein was separated on 12% Tris-Glycine-SDS polyacrylamide gels (Thermo Scientific) and transferred to nitrocellulose membrane. Membranes were blocked overnight at 4°C in 5% milk in TBST, and incubated with primary antibodies including mouse anti-Tau (Tau-5, 1:1000, Millipore), rabbit anti-Tau phospho-Threonine 231 (Tau-pT231, 1:1000, Millipore), and mouse anti-GAPDH (6C5, 1:5000, Advanced ImmunoChemical) for 2 h at room temperature. Membranes were then rinsed in TBST and incubated with appropriate HRP conjugated secondary antibody, either donkey anti-mouse or goat anti-rabbit (1:10,000, Santa Cruz Biotechnology), for 1 h at room temperature. Protein was detected using SuperSignal chemiluminescent substrate (Pierce Thermo Scientific) and quantified by ImageJ (NIH). Membranes were stained sequentially for Tau-5 and GAPDH, stripped in stripping buffer (2% SDS, 100 mM β-mercaptoethanol, 50 mM Tris, pH 6.8) at 50°C for 30 minutes, and stained for Tau-pT231. Results were replicated 3 times to ensure accuracy.
Forebrain hemispheres were sonicated in 0.2% diethylamine (DEA) in 50 mM NaCl with protease inhibitor (Sigma) at a volume of 1 ml per 100 mg of tissue. Samples were spun at 100,000 x g (53,000 rpm) for 30 minutes at 4°C and supernant collected, neutralized in 0.5 M Tris-HCl (pH 6.8), and analyzed by ELISA as previously described (Kawarabayashi et al., 2001
). The pellet was then sonicated in 70% formic acid, centrifuged, and supernant analyzed by ELISA. Mouse Aβ levels were analyzed in Kcna1
−/− and Kcna1
+/+ mice using capture antibody BNT77, which specifically recognized rodent Aβ11–28, and BA27 and BC05 antibodies to detect Aβ40
Data was analyzed for statistical significance using SPSS 16.0 (IBM). Survival analysis was completed by Kaplan-Meier log rank (Mantel-Cox). One-way ANOVA, with Tukey post hoc when necessary, was used to compare tau protein expression, abnormal EEG discharge duration, hippocampal volume, and forebrain volume between genotypes. Comparisons of seizure frequency between genotypes were made using the non-parametric Kruskal-Wallis test. Electrophysiology data was analyzed by One-way ANOVA with Bonferroni post hoc and Aβ level comparisons made by t-test (two-tailed) using GraphPad Prism 5.0 (Graphpad Software). All analysis of Drosophila results was completed by Chi-Squared analysis. Error bars represent standard deviation (SD) unless otherwise stated with the exception of in vitro electrophysiology results, which are reported as standard error of the mean (SEM).