Neuropsin-tTA heterozygous transgenic mice (Yasuda and Mayford, 2006
) were crossed with tet-APP heterozygous transgenic mice (Jankowsky et al., 2005
) to generate four genotypes: tTA/tet doubly transgenic (EC-APP) mice, tTA or tet singly transgenic mice, and NTG controls. Mice did not receive doxycycline so as to maintain expression of the tet-APP transgene. Neuropsin-tTA mice on the C57BL6 background were kindly provided by Dr. Mark Mayford. Tet-APP mice (Jackson Laboratory) were on a C57BL6/C3H background (stock #006004) or after backcrossing for >10 generations onto a C57BL6 background (stock #007049). Because C57BL6/C3H tet-APP mice were available first, most data were obtained on this background. However, soluble Aβ measurements in 3-month-old mice, detection of oligomers in 13-month-old mice, electrophysiological and EEG recordings were performed on the pure C57BL/6 background. Immunohistochemical and western blot analyses of EC-APP mice on the C57BL/6 or C57BL6/C3H background yielded comparable results (data not shown). All analyses were done in gender-balanced groups. The experimenters were blinded to genotype and treatment for these studies. The Institutional Animal Care and Use Committee of the University of California, San Francisco approved all experiments.
One cohort of mice was evaluated at 4, 9, and 13 months of age in the elevated plus maze and MWM. The same mice were analyzed in the Y-maze at 9 and 13 months.
Elevated Plus Maze
The elevated plus maze consisted of two open (without walls) and two enclosed (with walls) arms elevated 63 cm above the ground (Hamilton-Kinder, Poway, CA). Mice were allowed to habituate in the testing room under dim light for 1 h before testing. During testing, mice were placed at the junction between the open and closed arms of the plus maze and allowed to explore for 5 min. The maze was cleaned with 70% alcohol after testing of each mouse. Total distance traveled and time spent in the open and closed arms were calculated based on infrared photobeam breaks.
The apparatus consisted of three symmetrical arms in the shape of a Y. Before testing, mice were transferred to the testing room and acclimated for at least 1 h. During testing, each mouse was placed in a starting arm facing the wall and arm entries were recorded for 6 minutes, divided into six 1-min intervals. The maze was cleaned with 70% alcohol between testing of each mouse. Spontaneous alternations and total activity were calculated.
Morris Water Maze
The water maze consisted of a pool (122 cm diameter) filled with water (21±1°C) made opaque with nontoxic white powder. The pool was surrounded by distinct extra-maze (spatial) cues. Before hidden platform training, mice were given four pre-training trials in which they had to swim in a rectangular channel (15 cm × 122 cm) and mount a platform submerged 1.5 cm in the middle of the channel. Mice that did not mount the platform within 90 s were guided to it and allowed to sit on it for 10 s. The day after pre-training, mice were trained in the circular water maze.
For hidden platform training, the platform (14 cm × 14 cm) was submerged 1.5 cm. The platform location remained the same, but the drop location varied semi-randomly between trials. Mice that did not find the platform within 60 s were guided to it and allowed to sit on it for 10 s. At 4 and 9 months of age, mice received two training sessions with 3-h inter-session intervals for five consecutive days. Each session consisted of two trials with 10 min inter-trial intervals. At 13 months, mice were trained for only 3 days followed by reversal training (see below). Spatial probe trials, during which the platform was removed and mice were allowed to swim for 60 s before they were removed, were carried out 24 h after hidden platform training. For these trials, the drop location was 180° from where the platform was placed during hidden platform training.
For reversal training, the target platform was moved to the opposite quadrant 24 h after the probe trial and mice were trained to the new target location as above for three consecutive days. Another probe trial was given 24 h after the last hidden reversal session.
Behavior was recorded with a video tracking system (Noldus). Escape latencies, distance traveled, swim paths, swim speeds, percent time spent in each quadrant, and platform crossings were recorded for subsequent analysis. Thigmotaxis behavior was monitored during the last trial of hidden platform training in both initial acquisition and reversal-learning. No mice exhibited thigmotaxis in these experiments. Floating behavior was also monitored during training. Only one mouse (a singly tTA transgenic at 13 months) exhibited floating and was removed from the analysis.
γ-Secretase Inhibitor Treatment
NTG and EC-APP mice were injected subcutaneously once daily for 2 days with 3 mg/kg LY-411575 (a generous gift of Dr. J. Tung and the Myelin Repair Foundation, Saratoga, CA) or vehicle (corn oil). Six to 8 h after the last injection, mice were tested in the elevated plus maze. All mice were sacrificed directly after the elevated plus maze (approximately 10 h from the second LY-411575 injection), and brains were harvested and frozen for ELISA and Western blot analyses.
Mice were anesthetized with Avertin (250 mg/kg; 2,2,2-tribromoethanol dissolved in tert-amyl alcohol and PBS) and transcardially perfused with 0.9% saline. One hemibrain was drop-fixed in 4% paraformaldehyde for 48 h and the other hemibrain immediately frozen at −70°C. Coronal sections (30 μm) were prepared with a sliding microtome and collected for immunohistochemistry. Primary antibodies were rabbit anti-calbindin (1:20,000; Swant, Bellinzona, Switzerland), rabbit anti-Fos (1:10,000; Ab-5, Oncogene, San Diego, CA), rabbit anti-NPY (1:8,000; Immunostar, Hudson, WI), mouse biotinylated anti-Aβ (1:400; 3D6, Elan Pharmaceuticals, South San Francisco, CA), mouse anti-APP/Aβ (1:10,000; clone 6E10; Covance, Princeton, NJ) mouse anti-hAPP (1:2000, clone 8E5, Elan Pharmaceuticals), and mouse anti-synaptophysin (1:1000; Boehringer Mannheim, Indianapolis, IN). Sections labeled with anti-synaptophysin were incubated with FITC-conjugated horse anti-mouse IgG secondary antibody (1:75; Vector Laboratories, Burlingame, CA). Binding of other primary antibodies was detected with biotinylated donkey anti-rabbit or anti-mouse (1:1000; Jackson Immunoresearch Laboratories, West Grove, PA), followed by incubation with avidin-biotin complex (Vector Laboratories). Diaminobenzidine was used as the chromagen. Fos, calbindin, NPY, synaptophysin, and Aβ immunoreactivities were quantified in a behaviorally naive cohort of mice at 6 months and in mice that underwent behavioral testing at 13 months of age. Quantifications were performed as described (Mucke et al., 1995
; Palop et al., 2003
; Chin et al., 2004
; Chin et al., 2005
; Palop et al., 2005
; Palop et al., 2007
; Harris et al., 2010
Aβ ELISAs and Immunoblotting
For Aβ and APP measurements in specific regions, brain tissues were microdissected into DG, EC, CA, and RC. To obtain DG samples, the granule cell and molecular layers were microdissected away from the hilar region and CA3. For Aβ ELISAs, samples were snap-frozen and homogenized in 5 M guanidine buffer. Aβ1-x (approximates total Aβ) and Aβ1–42 were quantified as described (Johnson-Wood et al., 1997
; Mucke et al., 2000
). Low-molecular-weight oligomeric Aβ species were detected in microdissected DG, CA1, and EC samples homogenized in buffer containing 0.5 mM EDTA, 1 mM DTT, 0.1 M PMSF, Phosphatase Inhibitor Cocktails I and II (Sigma-Aldrich, St. Louis, MO), and protease inhibitors (Roche, Indianapolis, IN) in PBS. Samples (50 μg) were immunoprecipitated with anti-Aβ1–40 (5C3, 5 μl, Calbiochem, San Diego, CA) and Aβ1–42 (8G7, 5 μl, Calbiochem) antibodies overnight at 4°C. Dimers and trimers of Aβ were then measured by western blotting as described (Chin et al., 2005
; Harris et al., 2010
) with the additional step of microwaving the nitrocellulose membrane for 2.5 min before blocking in 3% BSA/Tris-buffered saline with 0.5% Tween and incubating with the primary anti-Aβ antibodies. The primary antibody cocktail consisted of 6E10 (1:1000), 4G8 (1:1000, Covance), and 82E1 (1:500, IBL-America, Minneapolis, MN) diluted in 3% BSA/Tris-buffered saline with 0.5% Tween. APP levels were assessed by western blotting in microdissected samples essentially as described (Chin et al., 2005
; Harris et al., 2010
) with mouse anti-APP/Aβ (1:1000, clone 6E10) and rabbit anti-GAPDH (1:10,000, Sigma-Aldrich) overnight at 4oC. Primary antibodies were detected with HRP-conjugated anti-mouse secondary antibody (1:10,000, Calbiochem) and bands were visualized by ECL. Densitometry measurements of the bands were acquired from scanned images with Quantity One software (Bio-Rad, Hercules, CA).
Mice were implanted for video EEG monitoring after anesthesia with intraperitoneal ketamine (75 mg/kg) and medetomidine (1 mg/kg). Teflon-coated silver wire electrodes (0.125-mm diameter) soldered to a multichannel electrical connector were implanted into the subdural space over the left frontal cortex (coordinates relative to bregma were: M/L, ±1, A/P ±1) and the left and right parietal cortex (M/L, ±2, A/P, −2). The left frontal cortex electrode was used as a reference. All EEG recordings were carried out at least 10 days after surgery on freely moving mice in a recording chamber. EEG activity was recorded with the Harmonie software (version 5.0b, Stellate, Canada) for 24 h. The number of sharp-wave discharges was automatically detected by the Gotman spike and seizure detectors (Harmonie, Stellate) and manually verified.
Electrophysiological Recordings in Acute Slices
At 6 and 14 months of age, NTG and EC-APP mice were anesthetized with Avertin or isoflurane and decapitated. The brains were quickly removed and placed in ice-cold solution containing (in mM) 2.5 KCl, 1.25 NaPO4, 10 MgSO4, 0.5 CaCl2, 26 NaHCO3, 11glucose, and 234 sucrose (pH ~7.4; 305 mOsmol). Coronal slices (400 μm) were cut on a Vibratome, collected in the above solution, and incubated for 30 min in standard artificial cerebrospinal fluid (ACSF; 30°C) containing (in mM): 2.5 KCl, 126 NaCl, 10 glucose, 1.25 NaH2PO4, 1 MgSO4, 2 CaCl2, and 26 NaHCO3 (290 mOsmol; when gassed with a mixture of 95% O2–5% CO2, the pH ~7.4). The slices were maintainedat room temperature for at least 30 min before recording. No recordings were made from slices more than 5 h after dissection. Individual slices were transferred to a submergedrecording chamber, where they were perfused with ACSF at a rate of 2 ml/min.
fEPSPs were recorded with glass electrodes (~3 MΩ tip resistance) filled with 1 M NaCl and 25 mM HEPES (pH, 7.3) and evoked every 20 s with a bipolar tungsten electrode (FHC, Bowdoin, ME). Recordings were filtered at 2 kHz (−3 dB, eight-pole Bessel), digitally sampled at 20 kHz with a Multiclamp 700A amplifier (Molecular Devices, Foster City, CA), and acquired with a Digidata-1322A digitizer and pClamp 9.2 software. In CA1, the stimulating electrode was placed in the stratum radiatum at the border of CA3 and CA1, and the recording electrode was placed ~150 μm away in CA1 stratum radiatum. In the DG, the stimulating electrode was placed in the medial perforant path (MPP) in the dorsal blade of the DG, and the recording electrode was also placed in the MPP ~150 μm closer to CA3 than the recording electrode. fEPSPs were recorded in the presence of 50 μM picrotoxin (Tocris). Synaptic transmission strength in the DG and CA1 were assessed by generating input-output (I-O) curves for fEPSPs. For each slice, we measured the fiber volley amplitude and initial slope of the fEPSP responses to a range of stimulation from 25 to 800 μA, and a response curve was generated for both values. Stimulus strength was then adjusted to be ~30% of the maximal fEPSP response for recordings that followed. After a 10 min stable baseline was established, LTP was induced in CA1 by high frequency stimulation (four 100-Hz trains of 100 stimuli every 20 s) and in the DG by theta burst stimulation (a set of 10 bursts repeated 10 times every 15 s; each burst, consisting of four pulses at 100 Hz, was repeated at 5 Hz).
For all data acquisition, experimenters were blinded with respect to the genotype and treatment of mice. Statistical analyses were conducted with GraphPad Prism version 4.0 or 5.0 (La Jolla, CA). Differences between means were analyzed by two-tailed Student’s t-test, one-way or two-way ANOVA with post-hoc tests as appropriate. Data were considered significant when p < 0.05.