Animals
JNPL3 (BL6) transgenic mice (Taconic, New York) were backcrossed in a C57BL/6 background (>10 generations). Age-matched control wild-type (WT) mice were purchased from Taconic. Mice were maintained on a 12:12 hour L:D schedule with food and water available ad libitum. Mice were tested at six, 12 or 18 months of age, depending on the experiments. Procedures were approved by the New York University School of Medicine Institutional Animal Care and Use Committee.
Brain fractionation protocol for Western blot analysis
Tau solubility was analyzed using a modified protocol from [31–33]. Briefly frozen cortex sections were homogenized without thawing in 5 X vol/wg of RIPA buffer (50 mM Tris–HCl, pH 7.4; 1% Nonidet P-40; 0.25% Na-deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM PMSF; 1 mM Na3VO4; 1 mM NaF; Complete protease inhibitor cocktail, Roche, IN, USA), with a mechanical homogenizer (TH; Omni International, USA), and centrifuged at 20,000 X g for 20 min at 4°C. An aliquot of the supernatant representing the total tau fraction was kept for protein quantification and western blot analysis. The rest of the supernatant was adjusted to 1% sarkosyl (N-lauroylsarcosine), incubated for 30 min at room temperature with constant rotating, and centrifuged at 100,000g for one hr at 20°C. After high speed centrifugation, the pellet was washed with 1% sarkosyl and centrifuged again at 100,000g for one h at 20°C. The post wash pellet containing sarkosyl-insoluble, aggregated tau was resuspended and analyzed by SDS-PAGE. Tau in the sarkosyl pellet has been shown by immuno-electron microscopy to be filamentous [32], and it is synonymous with that identified by immunohistochemistry in NFTs. All fractions were diluted in O+ buffer (62.5 mM Tris–HCl, pH 6.8; 10% glycerol; 5% 2-mercaptoethanol; 2.3% SDS; 1 mM EGTA; 1 mM EDTA; 1 mM PMSF; 1 mM Na3VO4; 1 mM NaF; Complete protease inhibitor cocktail, Roche), a modified O buffer, boiled for 5 min, and kept at −20°C. Depending on the antibody used, 10 to 20 μg of protein were analyzed by western blotting. Equal amount of protein (BCA assay, Promega) was loaded and the samples were electrophoresed on 10-12% SDS-PAGE gels and transferred to nitrocellulose membranes. All blots were blocked (5% nonfat milk and 0.1% Tween 20 in TBS) and then incubated with various primary antibodies overnight. Subsequently, the blots were washed and incubated for 2 h at room temperature with peroxidase-conjugated, goat anti-rabbit (Thermo Scientific) or anti-mouse IgG (1:2000; Jackson ImmunoResearch). Immunoreactive bands were visualized and analyzed by enhanced chemiluminescent reagent (Pierce ECL, Thermo Scientific) using a Fujifilm LAS4000 imaging system and the Multi Gauge software (Fujifilm Life Science). To compare the relative amount of tau protein, the densities of the immuno-reactive bands corresponding to phospho-tau were normalized and reported relative to the amounts of total tau protein or α-tubulin.
Electrophysiology
Transverse hippocampal slices (400 μm) were prepared from one hemisphere of age-matched mice (12–18 months or 6–7 months of age) using a vibratome (from part of the cohort, the other hemisphere was used to perform immunostaining or in situ hybridization, see below). Slices were maintained in oxygenated ACSF containing the following (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 25 D-glucose, 2 CaCl2, and 1 MgCl2 at room temperature. For electrophysiology experiments, slices were transferred to interface recording chambers (preheated to 30°C) perfused with oxygenated ACSF. Extracellular field EPSPs (fEPSPs) were evoked by stimulation of Schaffer collateral pathway afferents and were measured by recording in stratum radiatum of area CA1. In order to determine the response range for each hippocampal slice, the stimulus range was divided into 10 arbitrary units. The slices were stimulated at each level, and the fEPSP was recorded. In each recording, the fiber volley amplitude was as a measure of the input stimulation to the fEPSP. The range of input values and their respective output values (measured as the fEPSP slope) were plotted as a mean to characterize basal synaptic transmission in transgenic and wild-type mice across all experiments. Baseline responses were calculated using the stimulation intensity that elicited 40-50% of the maximal fEPSP response as determined by the input–output relationship. Paired-pulse facilitation (PPF), an assay of normal presynaptic function, was induced with two stimuli of equal intensity (same as baseline intensity) presented in rapid succession at variable interpulse intervals, ranging from 10 ms to 300 ms. PPF was measured by examining the ratio of the fEPSP slope in response to stimulus 2 and that of stimulus 1. Before LTP-inducing high-frequency stimulation (HFS), stable baseline synaptic transmission was established for 20–30 min with a stimulus intensity of 40–50% of the maximum fEPSP. Stimulus intensity of the HFS was matched to the intensity used in the baseline recordings. LTP was induced by either one or four trains (2 min intertrain interval) of 100 Hz HFS for 1 s. Data were collected and presented as the average slope of the fEPSP from six individual traces collected over 2 min and then normalized to baseline recordings of fEPSPs. Hippocampal slices from Tg and WT mice were prepared simultaneously and placed in a chamber outfitted with dual-recording equipment, thereby minimizing day-to-day variability in slice preparations and recordings. For zolpidem treatment, slices were incubated 20 min prior to HFS with a subthreshold concentration of zolpidem (1 μM). Student’s t-test, Repeated Measures ANOVA or N-way ANOVA (where appropriate) were used for electrophysiological data analysis with p < 0.05 as significance criteria.
In situ hybridization
Brains (one hemisphere from mice also used for electrophysiology) were fixed overnight in buffered 2% paraformaldehyde (PFA) at 4°C. Next day, hemispheres were stored in 20% glycerol, 2% DMSO in phosphate buffer (PB) until the brains were sliced as consecutive serial coronal sections (5 series, 50 μm thickness). Brain sections were stored in cryoprotectant (300 ml ethylene glycol, 550 ml PB, 300 g Sucrose, volume to 1000 ml with H2O; pH7.2) until used. Serial brain sections were used to perform in situ hybridizations as previously described in [34]. Briefly, RNA probes were prepared using dioxygenin (DIG) RNA labeling kits (Roche). Sections were postfixed in 4% PFA for 10 min followed by a wash in phosphate-buffered saline (PBS). Sections were treated with 1.5% H2O2 in methanol and rinsed in PBS. Then, sections were treated with 0.2 M HCl and washed in PBS. Proteinase K (Roche) digestion (20 μg/mL in PBS) was carried out followed by a wash in PBS, and the sections were refixed for 5 min in 4% PFA and washed with PBS. The sections were acetylated for 10 min (2.2 g triethanolamine hydrochloride (Sigma), 540 μL of 10 N NaOH (Fisher Scientific), 300 μL of acetic anhydride (Sigma) in 60 mL water) and washed in PBS. RNA probes, prepared at a dilution of 2 μL/mL in hybridization solution (50% formamide, 10% dextran sulfate, 1% 100× Denhart's, 250 μg/mL yeast tRNA, 0.3 M NaCl, 20 mM Tris–HCl, pH8, 5 mM EDTA, 10 mM NaPO4, 1% sarkosyl), were incubated at 80°C for 2 min. Thereafter, 500 μL of the probe mix was applied to the brain sections and incubated at 55°C overnight. The next day, sections were subjected to high stringency wash in pre-warmed 50% formamide, 2× SSC at 65°C. Next, the sections were rinsed in RNase buffer (0.5 M NaCl, 10 mM Tris–HCl, pH 7.5, 5 mM EDTA), followed by an RNaseA (Roche) treatment (20 μg/mL in RNase buffer) for 30 min and followed by a wash in RNase buffer, all at 37°C. The high stringency washes were repeated twice for 20 min each at 65°C, followed by a 15 min rinse in 2× SSC, then 0.1× SSC, both at 37°C. Sections were then washed in Wash Buffer (WB, 100 mM maleic acid, 150 mM NaCl, 0.5% Tween-20) and blocked with Blocking Buffer (1% Boehringer Manheim in WB) followed by incubation with anti-DIG-POD antibody (Roche) overnight at 4°C. Next day, slices were washed in WB and then incubated with Blocking Buffer 2 (0.5% Casein, 150 mM NaCl, 100 mM Tris, pH 7.5). Next biotinyl-tyramide was added to the sections, followed by washes in WB. Then sections were incubated with Streptavidin – AP for 1h at room temperature followed by 3 washes in WB and a wash in NTMT buffer (100 mM NaCl, 100 mM Tris–HCl, pH 9.5, 50 mM MgCl2, 0.1% Tween-20). The sections were then placed in a light-protected environment with approximately 400 μL of BM-purple AP substrate (Roche) until satisfactory staining was achieved. Finally, the sections were rinsed twice in PBS, coverslipped using Crystal mount aqueous mounting media (Sigma) and images were acquired using a Leica DM 5000B light microscope. Using Image J software (National Institutes of Health), regions of interests (ROI) were defined in all consecutive slices of one mouse. The size of ROI varied to some extent from anterior to posterior based on each individual slice. General criteria for the CA1 ROI were determined by boundaries starting at the CA2-CA1 border till the subiculum-CA1 boundary, including the stratum oriens and stratum radiatum. Within these ROIs, the number of positive cells was counted. The number of positive cells was corrected to the volume of the ROIs. All cell counts were made by observers blind to genotype.
Immunohistochemistry
For fluorescent immunostaining to count number of SST or PV positive cells from mice used for electrophysiology, consecutive serial brain sections (50 μm thick) of freshly fixed brain stored in cryoprotectant (see above), were washed in PBS for 3× 10 min. For blocking and permeabilization we used “staining buffer” containing 0.05 M Tris, 0.9% NaCl, 0.25% gelatin, and 0.5% Triton X-100, pH 7.4. Primary antibodies, rabbit anti-somatostatin (1:1000; Peninsula Laboratories/Bachem) and mouse anti-parvalbumin (1:500; Sigma-Aldrich) were diluted in staining buffer and incubated overnight at 4°C. The next day, the brain sections were washed in PBS and incubated with donkey anti-mouse Cy3 antibody (1:200; Jackson Immunoresearch) and donkey anti-rabbit Alexa 488 antibody (1:200; Jackson Immunoresearch), or with donkey anti-mouse Alexa 488 (1:200; Jackson Immunoresearch) and donkey anti-rabbit Cy3 antibody (1:200; Jackson Immunoresearch) diluted in staining buffer with Hoechst for one h at room temperature. Finally, the brain slices were washed in PBS and mounted in Mowiol mounting solution (Mowiol 4–88). In order to examine if pathological tau was expressed in GABAergic interneurons, two other aged JNPL3 (BL6) mice (age 13 months) were perfused with 0.9% saline, followed by perfusion of 4% paraformaldehyde (PFA) in PBS. Brains were fixed overnight in buffered 2% paraformaldehyde at 4°C. Next day, hemispheres were stored in 20% glycerol, 2% DMSO in phosphate buffer (PB) until the brains were sliced as consecutive serial coronal sections (5 series, 50 μm thickness). Brains slices were immunostained as described above and incubated with rabbit anti-somatostatin (1:1000; Peninsula Laboratories/Bachem) or rabbit anti-parvalbumin (1:1000, Swant) and with mouse anti-PHF1 (1:500; gift from Dr. Peter Davies) or mouse anti-MC1 (1:100; gift from Dr. Peter Davies). Images of brains were acquired using a Zeiss LSM510 confocal microscope. To quantify the number of GABAergic interneurons per hippocampal region, the complete hippocampus was imaged using Zeiss Zen2009 software. Next, Z-stacked images were stacked to a maximum intensity projections and ROIs were defined in all consecutive slices of one mouse, i.e. hippocampal CA1 and dentate gyrus (DG) in Image J. ROI varied from posterior to anterior. General criteria for the DG ROI were determined by the boundaries of the granular layers. Criteria for the CA1 ROIs were determined as described above. Within these ROIs, the number of positive cells was counted. The average of all positive cells was corrected to the volume of the ROIs.
For DAB staining, consecutive serial brain sections (50 μm thickness) stored in cryoprotectant, were washed in TBS (10 mM Tris, 140 mM NaCl, pH 7.4), then incubated with 3% H2O2/0.25% Triton X-100 (Sigma) for 30 min and followed by incubation in 5% milk in TBS. Brain slices were then incubated overnight with mouse anti-PHF1 (1:1000) or mouse anti-MC1 (1:100) in 5% milk in TBS. Slices were then washed in TBS +0.05% Triton-X100 (TBS-T), incubated with goat anti-mouse-Biotin (1:1000; M.O.M. kit, Vector laboratories) secondary antibody diluted in 20% Superblock (Pierce)/TBS-T for 2 hours, washed with TBS-T, and incubated with ABC reaction (M.O.M. kit) in Superblock in TBS-T. Brain slices were washed in 0.2 M sodium acetate and developed in DAB + nickel ammonium sulfate (17mg of DAB, 1.25 g Nickel ammonium sulfate in 50 ml of 0.2 M sodium acetate and 0.3% H2O2). Slices were washed in sodium acetate and TBS and mounted. Images of brains were acquired using a Leica DM 5000B light microscope.
Biotin surface labeling
Biotin surface labeling was performed as described in [35]. Briefly, hippocampal slices were generated as described for electrophysiology. Slices were maintained in oxygenated ACSF containing the following (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 25 D-glucose, 2 CaCl2, and 1 MgCl2 at 32°C for at least 1 h to recover. Then slices were treated with 0.5 mg/ml Sulfo-NHS-SS-biotin (Pierce) for 45 min on ice to label surface proteins. Slices were then washed in Tris-ACSF (25mM Tris pH 7.2 + ACSF). Slices were snap frozen and lysed in homogenization buffer (in mM: 40 HEPES pH 7.5, 150 NaCl, 10 pyrophosphate, 10 glycerophosphate, 1 EDTA) containing protease inhibitor and phosphatase inhibitor cocktail II and III (Sigma). Homogenates were cleared by centrifugation at 13000 rpm for 10 min at 4°C. Then samples were run on a SDS-page gel and immunoblotted against GABAAR alpha1 (1:1000, Neuromab), GABAAbeta2/beta3 (1:1000, Upstate) and GAPDH (1:10000, Cell Signaling).
Behavioral studies
Contextual fear conditioning
Tg and control mice were tested at the age of 5–6 or 12–13 months. Apparatus: Mice were trained and tested using the FreezeFrame system (Coulbourn Instruments). For training, mouse test cages equipped with stainless-steel shocking grids were connected to a precision feedback current-regulated shocker (Coulbourn Instruments). Each test cage was contained in a sound-attenuating enclosure (Coulbourn Instruments). Behavior was recorded using low-light video cameras. Stimulus presentation was automated using Actimetrics FreezeFrame software version 2.2 (Coulbourn Instruments). All equipment was thoroughly cleaned with water followed by isopropanol between sessions. Fear conditioning: Mice were habituated for 2 min on a shocking grid (context: shocking floor grids, vanilla scent). Fear conditioning was conducted with three 2 s, 0.5-mA footshocks (US) separated by 30 s. After conditioning, mice were returned to their home cages. Fear memory: Mice were retested in the training context (shocking grid, vanilla scent) 1 h 24 h and 7 days after training. Freezing behavior was measured using FreezeFrame (Coulbourn Instruments). Student’s t-test was used to analyze the data with p<0.05 as significant criteria.
Prepulse inhibition (PPI)
PPI test was used to study sensorimotor gating. Tg and control mice were tested at the age of 5–6 or 12–13 months. The testing apparatus of a startle response system was contained in a sound attenuating chamber calibrated for responses from mice that are 20–35 g in weight (San Diego Instruments, San Diego, USA). Each mouse was placed in a clear, cylindrical holding tube within a sound-attenuating chamber and habituated for 4 min immediately prior to testing. The test started with 5 startle pulses of 120 dB to measure startle responses. This was followed by 5 blocks of randomized trials: no stimulus, startle stimulus (120 dB), 72 dB prepulse+120 dB startle, 84 dB prepulse+120 dB startle, 90 dB prepulse+120 dB startle. The test was finalized by 5 startle pulses of 120 dB to measure habituation. The prepulses were presented 100 ms before startle stimulus. The inter-block interval ranged from 6–20 s. All tests were performed at the same time of day following identical habituation periods. Mice failing to demonstrate acoustic startle response at 2,5× baseline were excluded as hearing impaired. Responses were detected as changes within the holding tube. Student’s t-test was used to analyze the data with p<0.05 as significant criteria.
Hotplate
The hotplate test was used to determine any differences in nociceptive responses. Mice were placed on a hotplate preheated to 50°C (Columbus Instruments). The animal was observed and the time for the animal to lift one of its hind paws was recorded. The mice were then immediately removed from the hotplate and placed back in their cage. The hind paw is used to determine nociception because lifting the front paws is also normal exploratory behavior. Student’s t-test was used to analyze the data with p<0.05 as significant criteria.